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+    "id": "PMC2527523",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2527523\nAUTHORS: Satoshi Naganawa, Masaru Yokoyama, Teiichiro Shiino, Takeyuki Suzuki, Yoshiaki Ishigatsubo, Atsuhisa Ueda, Akira Shirai, Mitsuhiro Takeno, Satoshi Hayakawa, Shigehiro Sato, Osamu Tochikubo, Shingo Kiyoura, Kaori Sawada, Takashi Ikegami, Tadahito Kanda, Katsuhiko Kitamura, Hironori Sato\n\nABSTRACT:\nThe third variable region (V3) of the human immunodeficiency virus type 1 (HIV-1) envelope gp120 subunit participates in determination of viral infection coreceptor tropism and host humoral immune responses. Positive charge of the V3 plays a key role in determining viral coreceptor tropism. Here, we examined by bioinformatics, experimental, and protein modelling approaches whether the net positive charge of V3 sequence regulates viral sensitivity to humoral immunity. We chose HIV-1 CRF01_AE strain as a model virus to address the question. Diversity analyses using CRF01_AE V3 sequences from 37 countries during 1984 and 2005 (n = 1361) revealed that reduction in the V3's net positive charge makes V3 less variable due to limited positive selection. Consistently, neutralization assay using CRF01_AE V3 recombinant viruses (n = 30) showed that the reduction in the V3's net positive charge rendered HIV-1 less sensitive to neutralization by the blood anti-V3 antibodies. The especially neutralization resistant V3 sequences were the particular subset of the CCR5-tropic V3 sequences with net positive charges of +2 to +4. Molecular dynamics simulation of the gp120 monomers showed that the V3's net positive charge regulates the V3 configuration. This and reported gp120 structural data predict a less-exposed V3 with a reduced net positive charge in the native gp120 trimer context. Taken together, these data suggest a key role of the V3's net positive charge in the immunological escape and coreceptor tropism evolution of HIV-1 CRF01_AE in vivo. The findings have molecular implications for the adaptive evolution and vaccine design of HIV-1.\n\nBODY:\nIntroductionThe third variable region (V3) of human immunodeficiency virus type 1 (HIV-1) envelope gp120 subunit participates in determination of viral infection coreceptor tropism [1], [2]. It is usually composed of 35 amino acids, which form a loop-like structure on the gp120 monomer [3], [4]. The V3 and the conserved outer domain of gp120 create the binding surface for viral infection coreceptors after the binding of gp120 to the primary infection receptor CD4 [4], [5]. These interactions and successive conformational changes of gp120 are essential in rendering the initially occluded hydrophobic domain of the envelope gp41 subunit available to fusion with cellular plasma membrane.The HIV-1 V3 is highly variable. In parallel with the V3 sequence variation, many types of infection coreceptors are reported. These are the members of the G protein-coupled receptor superfamily. The two most common types of infection coreceptors in humans are the CC chemokine receptor 5 (CCR5) and the CXC chemokine receptor 4 (CXCR4) [6]. Notably, a single group of the HIV-1 variants using the CCR5 (R5 virus [6]) predominates during the first several to 10 years or more of persistent infection in vivo\n[7], [8]. Other tropism variants including CXCR4-tropic variants (X4 virus [6]) can grow at early stage of infection by needle stick injuries, but are replaced with the R5 viruses after seroconversion [9], [10]. They generally grow better only during progression to AIDS. The R5 and X4 viruses are distinguishable by sequence feature of V3: the R5 V3 amino acid sequences generally have a lower net positive charge than those of X4 [3], [11]. Only a few basic substitutions in V3 can switch the viral coreceptor tropism from CCR5 to CXCR4 [12], [13]. Considering the extremely high levels of mutation rate of HIV-1, these findings suggest that strong selective forces are continually purifying the R5 viruses during long-lasting persistent infection.The HIV-1 V3 is highly immunogenic, tolerant to change, and variable presumably to evade immune recognition [14]–[16]. HIV-infected individuals make high levels of anti-V3 antibodies that are reactive with soluble, monomeric gp120 protein [17], [18]. However, they often react poorly or only with low affinity to the native, oligomeric form of the gp120 protein [17], [18]. The inaccessibility of the oligomeric envelope protein is particularly prominent in the primary HIV-1 isolates [19]–[21], which are usually the R5 viruses. Indeed, studies with limited set of viruses have shown that antibodies reactive with the R5-virus V3s tend to bind to the monomeric but not the oligomeric gp120s [22], [23], and they poorly neutralize the R5 viruses [23]–[25]. In contrast, antibodies against X4-virus V3s usually bind to both forms of gp120s [22], [23], and they potently neutralize the X4 viruses [23]–[25]. Consistent with the lower sensitivity of R5 viruses to anti-V3 antibody neutralization, positive selection for amino acid variation is less prominent in the R5 virus V3 sequences, and V3 amino acid sequences of the R5 virus are relatively homogeneous among virus isolates [26], [27] or in infected individuals [28]–[30] compared with those of the X4 viruses.While the immunological escape, variation, and coreceptor tropism evolution of HIV-1 is an important issue from both clinical and scientific viewpoints, current studies are largely confined to those of HIV-1 subtype B from North America and Europe. In this study, we attempted to obtain and integrate information on HIV-1 CRF01_AE strain [31] circulating in Southeast Asia. Specifically, we examined whether net positive charge of HIV-1 CRF01_AE V3 sequence regulates viral sensitivity to humoral immunity. Here, we demonstrate by combining bioinformatics, experimental, and protein modelling approaches that the reduction in net positive charge of HIV-1 CRF01_AE V3 sequence reduces viral sensitivity to humoral immunity and simultaneously confers viral CCR5 tropism. The findings suggest a key role of the V3 net positive charge in the immunological escape, variation, and the coreceptor-tropism evolution of HIV-1 CRF01_AE in vivo.ResultsCorrelation of HIV-1 CRF01_AE V3 net positive charge, V3 prevalence, and V3 diversityA previous case study has suggested that a group of CRF01_AE V3 sequences for the viral CCR5 tropism is resistant to the selective force for amino acid variation [29], [30]. To extend this finding with three infected individuals, we conducted large-scale analysis of V3 diversity using public database information. V3 sequences of the CRF01_AE strain were extracted from the HIV Sequence Database (http://www.hiv.lanl.gov/content/hiv-db/mainpage.html). A single V3 amino acid sequence per infected individual was randomly extracted. Sequences with ambiguous bases are excluded from the analysis. V3 sequences (n = 1361, 35 amino acid length) from 37 countries during 1984 and 2005 (see supporting information Figs. S1a and S1b) were used for the diversity analysis. The 1361 V3 sequences were divided into two subsets, “a” and “b”, which lack and have the glycosylation motif, respectively. Each group was divided into subgroups on the basis of the net charge; arginine, lysine, and histidine were counted as +1, aspartic acid and glutamic acid as −1, and other amino acids as 0.Although there are exceptions, the V3 amino acid sequences capable of directing the viral CCR5 tropism of the CRF01_AE strain generally have net positive charges of +2 to +4 and the conserved N-linked glycosylation motif (asparagine-X-threonine/serine) at positions 6 to 8 [29], [30]. Consistent with the dominance of the R5 viruses in humans, less positively charged, glycosylated V3 sequences for the CCR5 tropism (2b, 3b, and 4b) were dominant in the database for over 15 years, independent of the sampling period (Fig. 1A and Fig. S1c). Shannon entropy scores representing amino acid variation were relatively low for the most abundant 3b V3 compared to those for the 7a V3 for the CXCR4 tropism (Fig. 1B), consistent with previous report [3]. Nucleotide substitutions for amino acid change were more suppressed in the V3s for the CCR5 tropism compared with those for the CXCR4 tropism (Fig. 1C). The 3b V3 had the lowest ratio of nonsynonymous to synonymous substitutions (d\nn/d\ns) with about 0.6, and acquisition of a glycosylation site decreased the d\nn/d\ns ratios (P = 0.001, Table S1). The d\nn/d\ns ratios correlated positively with the Shannon entropies, with lower d\nn/d\ns ratios for lower entropies (Fig. 1D). Similar effects of the net positive charge of V3 on V3 diversity were detected in other major genetic lineages of HIV-1 circulating in the world, such as subtypes A, B, and C (Fig. S2).10.1371/journal.pone.0003206.g001Figure 1V3 net positive charge influences V3 diversity.HIV-1 CRF01_AE V3 sequences (n = 1361) were grouped on the basis of net positive charge and glycosylation capability (A) Distribution of the V3 structural variants of HIV-1 CRF01_AE in the public database. (B) Shannon entropy scores [3] on primary and three-dimensional structures of 3b and 7a V3s. AE-c, 3b-c, and 7a-c indicate consensus sequences for all CRF01_AE sequences, 3b V3 group (n = 576), and 7a V3 group (n = 21), respectively. (C) Median (diamond) and interquartile range (vertical bar) of ratios of d\nn/d\ns, (D) Relation of median d\nn/d\ns ratios and average Shannon entropy scores, and (E) Tajima's D statistic values [32] for each V3 structural group.If the low levels of amino acid changes in the V3 structures for CCR5 tropism involved the elimination of new mutants in natural selection, negative values for Tajima's D statistic would be expected [32]. Indeed, Tajima's D statistic was significantly negative for 2b and 3b V3s (P = 0.01, Fig. 1E and Table S2). Together, these findings on V3 diversity provide further evidence that the V3 sequences for the CCR5-tropism are less variable in nature due to the limited positive selection for amino acid diversity compared with those for CXCR4 tropism.Correlation of HIV-1 CRF01_AE V3 net positive charge, HIV-1 neutralization sensitivity, and HIV-1 coreceptor tropismA positive selection pressure for the V3 diversity can be the humoral immunity. To examine whether the V3 net positive charge regulates HIV-1 neutralization sensitivities to the anti-V3 antibodies, we used V3 recombinant viruses (n = 30). The recombinant viruses have the CRF01_AE V3s in the backbone of the X4 virus gp120 of HIV-1 subtype B, LAI strain [13], [30], [33]. The V3s were from HIV-1 proviral DNA clones in the peripheral blood mononuclear cells of three infected individuals at the asymptomatic stage or AIDS [13], [30], [33]. These V3s could be grouped into the 2b (n = 2), 3b (n = 4), 4b (n = 5), 5b (n = 3), 6b (n = 5), 3a (n = 1), 4a (n = 2), 5a (n = 3), 6a (n = 3), and 7a (n = 2) sequences (Fig. S3). The 2b, 3b, and 4b V3 clones were the most prevalent in the three infected individuals examined, and their sequences were mostly identical [29], consistent with the V3 prevalence and diversity data in this study. The neutralization sensitivity of each recombinant virus was assessed with a single-round viral infectivity assay [34]. In parallel, titers of plasma antibody reactive with the V3 elements of the recombinant viruses were measured with V3-peptide-based, enzyme-linked immunosorbent assay (ELISA) [35].When the V3 synthetic peptide of the parental virus of the recombinant viruses was used for the immunoassay, the CRF01_AE plasma samples had only traces of binding antibodies (Fig. 2B, Absorbance of LAI). The blood samples failed to neutralize this virus (Fig. 2B, ND50 of LAI). Thus, the blood samples tested are lacking in anti-V3 binding antibodies, as well as neutralization antibodies against the parental subtype B virus. The results agree with low levels of V3 amino acid identities between subtype B and CRF01_AE strains (http://www.hiv.lanl.gov/content/hiv-db/mainpage.html). They also agree with the assumption that V3 sequence diversity causes neutralization escape of HIV-1.10.1371/journal.pone.0003206.g002Figure 2Reduction in V3 net positive charge causes loss of HIV-1 neutralization sensitivity to blood antibodies against V3 PND.\n(A) Genome structure of the V3 recombinant viruses (n = 30) [13], [30], [33]. (B) Blood anti-V3 antibody titers and viral neutralization sensitivity to the blood antibodies. Plasma samples (n = 20) were obtained from CRF01_AE positive individuals. Plasma antibody binding activities to the synthetic peptides corresponding to each V3 PND of the recombinant viruses were measured by V3-peptide-based ELISA [35] (Absorbance at 450 nm). The same plasma samples were used to measure ND50 against recombinant viruses in a single-round infectivity assay using CD4+CXCR4+CCR5+ HeLa cells [34] (Neutralization). Medians (diamond) and interquartile ranges for individual V3 structural groups are shown. (C) Neutralization sensitivity and coreceptor tropism. The recombinant viruses were grouped into CCR5-tropic (left) and CXCR4-tropic (right) variants using data reported previously [13], [30], [33].All blood samples from CRF01_AE infected individuals contained antibodies that bound to the synthetic peptides from the CRF01_AE strain V3 sequences of the recombinant viruses (Fig. 2B, Absorbance of 2b to 7a). Coincidentally, they neutralized a group of the viruses having particular V3 sequences (Fig. 2B). The neutralization-sensitive viruses had V3s lacking a glycosylation site (3a, 4a, 5a, 6a, 7a, n = 11), or V3s having a glycosylation site and increased net positive charge (5b and 6b, n = 8) (Fig. S3). The neutralization activities were abrogated by protein G pre-treatment of the plasma samples (Fig. S4), showing that neutralization is indeed mediated by antibodies in the plasma samples.Notably, the blood samples poorly neutralized a group of V3 recombinant viruses (Fig. 2B, ND50 of 2b, 3b, and 4b, n = 11). These viruses had weakly charged V3s and had an N-glycosylation site, which are the characteristics of V3s for CCR5 tropism (Fig. S3, V3 IDs of 2b, 3b, and 4b). The lack of neutralization activities was not due to the lack of anti-V3 binding antibodies against these recombinant viruses. The blood samples contained high levels of antibodies that bound to the 2b, 3b, and 4b V3 peptides, more so than of other V3 groups (Fig. S5 and Fig. 2B, Absorbance of 2b, 3b, and 4b). The results are consistent with the high levels of prevalence and limited diversity of these V3 sequences (Fig. 1). The study shows that the group of V3 elements for CCR5 tropism is highly immunogenic, whereas binding antibodies raised in humans generally show only weak neutralization activities. This neutralization-resistant phenotype associated with particular V3 group was observed reproducibly in a multiple-round infectivity assay, suggesting that the phenotype is intrinsic to the viruses (Fig. S6).The relation of this neutralization-resistant phenotype and HIV-1 coreceptor tropism was examined using information on coreceptor usages of the V3 recombinant viruses [13], [30], [33]. Importantly, the V3s having the viral-resistant phenotype unexceptionally rendered HIV-1 CCR5-tropic (Fig. 2C, left panel, V3 groups of 2b, 3b, and 4b, n = 10). On the other hand, V3s conferring CCR5 tropism did not always render HIV-1 resistant (Fig. 2C, V3 groups of 3a, 4a, 5a, and 5b, n = 7). Thus, the V3s associated with the neutralization resistance are a subset of the V3 elements associated with viral CCR5 tropism. In contrast, all V3s associated with viral CXCR4 tropism and CCR5/CXCR4 dual tropism rendered HIV-1 susceptible to neutralization (Fig. 2C, V3 groups of 4b, 5a, 5b, 6a, 6b, and 7a, n = 13).The group of viruses having 4b V3s were neutralization-resistant only when they had the CCR5-restricted tropism (Fig. 2C, 4b in left and right panels). The 4b V3s associated with viral neutralization resistance had no basic substitutions compared with the CRF01_AE consensus (Fig. S3, recombinant IDs of A1, A2, A4, and A5). By contrast, the neutralization-sensitive version had two basic substitutions (Fig. S3, recombinant ID of B10). The results may imply that V3 basic substitutions at particular positions in addition to the overall net positive charge play a critical role in the determination of viral neutralization sensitivity and coreceptor tropism.We further examined whether a CCR5-tropic but not a CXCR4-tropic envelope of CRF01_AE strain is linked to viral resistant to neutralization by anti-V3 antibodies. For this purpose, we used a pair of nearly isogenic R5 and X4 virus clones that have 3b and 5a V3, respectively [36], [37]. The results obtained with these clones and 35 blood specimens were consistent with present study and indicated that the CCR5-tropic but not the CXCR4-tropic envelope protein of the HIV-1 CRF01_AE strain was linked to viral resistance to neutralization with anti-V3 antibodies in the blood (data not shown).HIV-1 V3 net positive charge and V3 conformationTo obtain molecular insights into the roles of the V3 net positive charge in regulating HIV-1 neutralization sensitivity, we conducted computer-aided structural analysis. Currently, X-ray structure information on the HIV-1 R5 virus gp120 monomer bound with soluble CD4 [3] is available in the Protein Data Bank. With the data, we attempted to obtain a gp120 monomer structure for the pre-CD4 binding stage to address initial V3 conformation before receptor interaction. We first constructed gp120 outer domain models of the V3 recombinant viruses used in this study by a homology modelling method. Molecular dynamics (MD) simulation was then performed with the homology models.\nFigure 3A shows examples of the MD simulation of two recombinant virus gp120s with 3b and 7a V3 elements. The TH09 V3 (3b V3) is from an HIV-1 CRF01_AE infected asymptomatic patient, identical to the CRF01_AE V3 consensus sequence (Fig. S3, recombinant ID of TH09), rendered HIV-1 neutralization-resistant and CCR5-tropic (Fig. 2C, left panel). The B1 V3 (7a V3) is from an AIDS patient, more positively charged (Fig. S3, recombinant ID of B1) and rendered HIV-1 neutralization-sensitive and CXCR4-tropic (Fig. 2C, right panel).10.1371/journal.pone.0003206.g003Figure 3MD simulation of the HIV-1 gp120 outer domain.The V3 subset conferring the neutralization-resistant phenotype is referred to in this study as rV3: it has net positive charges of +2 to +4, an N-glycosylation site, and a capability to direct viral CCR5 tropism. The non-rV3 renders HIV-1 more susceptible to blood antibody neutralization. It has net positive charges of greater than +4 and a capability to direct viral CXCR4 tropism. (A) Examples of the MD simulation of two recombinant virus p120 outer domains with rV3 (TH09) and non-rV3 (B1). Distance between the Cα atom of P318 at the V3 tip and the Cα atom of Q433 at the β20β21 loop were monitored for 5 nanoseconds. (B) Superimposition of the gp120 monomers with the TH09 V3 (blue) or B1 V3 (red) at the simulation time of 5 nanoseconds. (C) Close-up view of the base-stem region of the TH09 V3. Orange dotted lines around the tip of the orange arrow indicate three hydrogen bonds at the V3 base. (D) Shannon entropy scores of the amino acids at the positions of 330, 332, and 424 in the public database. The positions in the gp120 of the HIV-1LAI\n[48] are used for the amino acid numbering.The MD simulations show that V3 configuration is nearly equilibrated up to 5 nanoseconds of simulation times (Fig. 3A). Notably, the TH09 V3 was equilibrated at a much more distant position from the β20β21 loop in the outer domain than the B1 V3 (Figs. 3A and B). Hydrogen bonds were formed around the TH09 V3 base between D330 and R332, and D330 and R424, which contributed to stabilizing the V3 configuration (Fig. 3C). However, the hydrogen bonds were not formed with the gp120 having the B1 V3 (Fig. S7). Coulombic repulsion between B1 V3 and R424 increased about 44-fold as compared with that of TH09 V3, with electrostatic energies of +2.0 and +0.045 kcal/mole for B1 and TH09, respectively. The repulsion was greatest on the R424 residue in the gp120 outer domain. The results suggest that an increase in the V3 net positive charge influences electrostatic balance at the V3 base.Importantly, the amino acids around the V3 base are relatively conserved in nature. The D330 and R332 are located at the V3 base and highly conserved within each subtype of the HIV-1 M group in the public database (Fig. 3D). The conservation was seen even in the V3s for the CXCR4 tropism (Fig. 1B). The R424 is in the fourth constant region (C4) of the gp120 core, and neighbouring amino acids are also conserved. The CRF01_AE strain alone has lysine at position 424, whereas K424 is conserved within the CRF01_AE. These data suggest that most HIV-1 gp120 monomers have the potential to stabilize V3 configuration at the base and that basic amino acid substitutions in V3 have strong influences on the V3 configuration.The MD simulation data were incorporated into those of a gp120 trimer structure obtained by cryoelectron microscopy [38] to illustrate schematically the V3 position in the native gp120 trimer (Fig. 4). Some glycans are also schematically illustrated at the appropriate regions. The models predict that less positively charged V3 protrudes into the outer domain of the neighboring gp120 monomer, whereas V3 with increased net positive charge protrudes away from the neighboring monomer in the trimer context.10.1371/journal.pone.0003206.g004Figure 4Models for the self-directed masking of V3 by mutations for the CCR5 tropism.The MD data in Fig. 3 and the HIV-1 gp120 trimer structure from cryoelectron microscopy [38] were used to construct the gp120 trimer models with CCR5-tropic (left) or CXCR4-tropic (right) V3. The models were made so that the MD data and experimental data [3], [4], [38] are compatible.DiscussionHIV-1 is the causative agent of AIDS and is responsible for more than 2 million deaths every year. Understanding the immunological escape, variation, and coreceptor tropism evolution of HIV-1 is critical for developing strategies for anti-HIV interventions. In this regard, current studies are largely confined to those of HIV-1 subtype B from North America and Europe. In this study, we focused on the study of HIV-1 CRF01_AE strain circulating in Southeast Asia. The HIV-1 CRF01_AE is one of the five major HIV-1 subtypes circulating in the world [31] and thus an important strain for public health of Asia, as well as of world. However, much less basic information are available as compared with the HIV-1 subtype B.We first demonstrate with bioinformatics approach using 1361 sequences in public database that CRF01_AE V3's net positive charge influences V3 diversity and prevalence. We found that the net positive charge of V3 influences V3 diversity (Fig. 1). Acquisition of the N-glycosylation motif in V3 augmented the sequence conservation. Our data of d\nn/d\ns ratios and Tajima's D statistic provide strong evidence that the reduction in V3 diversity is due to limited positive selection for amino acid changes. These findings are compatible with previous findings with subtype B that sequence diversity is smaller among R5 virus V3s [26]–[28]. Moreover, the findings demonstrate the generality of findings obtained with an intra-familial infection case of CRF01_AE infection [29], [30]. The evidence that the V3 net positive charge influences V3 diversity is not solely based on sequence analysis. Structural and neutralization data suggest that V3 net positive charge regulates V3 diversity by controlling V3 structure and neutralization sensitivities, as discussed below.We next examined potential causes of the differential diversity of CRF01_AE V3 sequences. We demonstrated with neutralization assay of V3 recombinant viruses that the V3 net positive charge influences HIV-1 neutralization sensitivity. We found that reduction in the net positive charge of V3 caused reduction in viral neutralization sensitivity to the blood anti-V3 antibodies in infected humans (Fig. 2). Again, acquisition of the N-glycosylation motif in V3 augmented the effect. We further confirmed that the especially neutralization resistant V3 sequences all render HIV-1 CCR5 tropic (Fig. 2C). These results are compatible with previous findings with subtype B that R5 viruses are more refractory to anti-V3 antibodies [23]–[25]. Together with sequence analysis data, these findings suggest that anti-V3 antibodies can act as a positive selection pressure to increase V3 sequence diversity and that V3 positive net charge can influence V3 diversity by regulating neutralization sensitivity to the V3 antibodies.Importantly, present data revealed that not all the CCR5-tropic V3 sequences render HIV-1 resistant to the neutralization. Some V3 sequences lacking N-glycosylation site or those have the glycosylation site but have net positive charge of +5 conferred CCR5 tropism on HIV-1, whereas they were relatively sensitive to the antibody neutralization (Fig. 2C). These data are consistent with findings on CRF01_AE V3 sequence diversity and prevalence in this study. Together, the data suggest that a particular subset of CRF01_AE V3 for CCR5 tropism confers a selective advantage on HIV-1 in the face of humoral immunity and that the anti-V3 antibodies may be an important selective force to maintain CCR5-tropic V3 sequences with limited amino acid changes during persistent infection.We further examined molecular mechanisms of neutralization escape. We found with MD simulation that reductions in the net positive charge of V3 caused a shift of V3 position in the gp120 monomer (Fig. 3). This is the first indication that the net positive charge of V3 regulates V3 configuration in the gp120 monomer. By incorporating our MD data into experimental data of HIV-1 gp120 structures [3], [4], [38], we proposed a model of V3 masking that explains how the net positive charge of V3 regulates HIV-1 V3 neutralization sensitivity (Fig. 4). The model explains present diversity and neutralization data: it predicts that less positively charged V3 for CCR5 tropism positions in the gp120 trimer context such that it protrudes into the outer domain of the neighboring gp120 monomer, which will inevitably result in better protection of V3 from antibodies by the glycans. This model also explains why anti-V3 antibodies bind effectively to monomeric but not native, oligomeric form of the gp120 protein of the HIV-1 R5 viruses [22]–[25]. Further structural studies are under way to assess the model.Our study provides molecular insights into the mechanisms of coreceptor-tropism evolution of HIV-1. Due to high levels of viral mutation rate, vigorous and continual viral replication in vivo, and viral tolerance to V3 mutations, many viable HIV-1 V3 mutants would be continuously generated during a persistent infection. Therefore, why only the R5 virus dominates during persistent infection is a long-lasting question in HIV-1 research. Our study suggests a selective advantage of a subset of CCR5-topic V3 sequences in the face of humoral immunity: self-masking of neutralization epitope by reduction in net positive charge. Higher levels of the d\nn/d\ns ratios for more positively charged V3 sequences for CXCR4 tropism (Figs. 1C and D) may imply that the X4 viruses might persist as a minority, continually receive positive selection pressures for amino acid changes, and outgrow only when the host immunity is severely damaged.It will be important to examine why neutralization resistant V3 sequences exclusively direct viral CCR5 usage (Fig. 2C). The distinct V3 configuration in gp120 may contribute to the restriction of coreceptor type to be interacted, because amino acid residues at the V3 base directly participate in the binding to the N-terminal region of CCR5 [4]. In addition, structural differences among chemokine receptors may contribute. For example, the N-terminal region of CXCR4 is glycosylated, whereas that of CCR5 is not. The glycosylated V3 for neutralization resistance will sterically interrupt the access to the glycosylated coreceptor. Indeed, the removal of the N-linked glycosylation sites from CXCR4 allows the protein to serve as a universal coreceptor for both X4 and R5 viruses [39]. Further structural studies are under way to address this issue.Our study has implications for HIV-1 vaccine design. The data suggests that a key impediment to the clinical use of Gp120 as an immunogen may be the cryptic nature of the R5 virus V3 neutralization epitope. A simple strategy to use R5 virus Gp120 will be insufficient even if the Gp120 of circulating HIV-1 subtype is carefully selected as immunogen. To develop strategies that could circumvent or overcome the impediment may be critical for practical application of Gp120 vaccine. In this regard, the amino acids that contribute to forming V3 conformation for the epitope masking through hydrogen bonds are relatively conserved among HIV-1 major subtypes in the world (Fig. 3D). Therefore, intervention in these interactions might be a target for a new strategy to improve effectiveness of immunological control of HIV-1.In conclusion, we have identified here structural and functional features of HIV-1 CRF01_AE V3 elements those allow HIV-1 less sensitive to antibody neutralization. To our knowledge, this is the first report to show that the net positive charge of a neutralization epitope regulates viral sensitivity to humoral immunity. Thus, amino acid substitutions altering charged status of antigen site appear to deserve more attention, particularly in the adaptive evolution of HIV-1, as well as the other rapidly evolving pathogen.Materials and MethodsAnalysis of sequence diversityGrouping of the sequences was done computationally using a software system, InforSense BioSense V3 (InforSense Ltd. http://www.inforsense.com). Nonsynonymous and synonymous nucleotide substitutions were calculated for all pair-wise sequence comparisons within each V3 subgroup using the Perl version of SNAP (Los Alamos HIV sequence database) according to the Nei and Gojobori method [40] incorporating the statistical method developed by Ota and Nei [41]. Amino acid variation at individual V3 positions was calculated according to the method described in the report by Huang et al [3] on the basis of Shannon's equation [42]:where H(i), p(xi), and i indicate the amino acid entropy score of a given position, the probability of occurrence of a given amino acid at the position, and the number of the position, respectively. An H(i) score of zero indicates absolute conservation, whereas 4.4 indicates complete randomness. The H(i) scores were expressed in the V3 sequence or in the three-dimensional structures constructed by the MD simulation method described below. The p(xi) scores were used to construct a consensus for each V3 structural group. Tajima's D statistic [32] for each type of V3 sequence population was calculated using DnaSP 4.10 [43].Blood specimensPlasma samples were obtained from HIV-1 CRF01_AE positive individuals with written informed consent at Yokohama City University Hospital in Japan according to the rule of the ethics committee of the hospital. The clinical stages of the patients at the time of blood collection were A1 (n = 4), A2 (n = 4), B3 (n = 3), C2 (n = 1), C3 (n = 7), and unknown (n = 1) according to the 1993 Revised Classification System (CDC, USA). The CD4+ T-cell counts and HIV-1 RNA levels ranged from 2×103 to 3719×103 /ml blood (mean = 243×103 /ml) and from <50 to 7.5×105 copies/ml blood (mean = 2.4×104 copies/ml), respectively. All plasma samples were heat-inactivated at 56°C for 30 minutes prior to use.Anti-V3 antibody titrationV3-peptide-based ELISA [35] was carried out using synthetic peptides matching to the central 19 amino acids of the V3 regions of the recombinant viruses (Fig. S3). Synthetic peptides were coated on 96-well plates (Immulon II; Dynatech Laboratories, Virginia, USA) and reacted with diluted plasma samples (1/104). Antibodies bound to the peptides were detected with anti-human IgG peroxidase conjugate and 3,3′,5,5′-tetramethylbenzidine substrate (TMB peroxidase EIA substrate Kit, Bio-Rad Laboratories, USA). Each plasma sample was tested in duplicate.Neutralization assaysPlasmid DNAs containing HIV-1 V3 recombinant proviruses (n = 30) were constructed by the overlap extension method [13], [30], [33]. Cell-free viruses were prepared by transfection of the plasmid DNAs into HeLa cells as described previously [13], [30], [33]. Neutralization activities were measured in a single-round viral infectivity assay using CD4+CXCR4+CCR5+ HeLa cells [34]. Equal infectious titers of viruses (300 blue-cell-forming units) were incubated with serially diluted plasma samples (1/10 to 1/103) for 60 min at 37°C. The infected cells were cultured for 48 hours at 37°C, fixed, and stained with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside. Each plasma dilution was tested in duplicate, and the means of the positive blue cell numbers were used to calculate the 50% inhibition dose of viral infectivity (ND50). For plasma samples that did not neutralize a virus at the lowest dilution tested (1:10), an arbitrary titer of 1:5 (50 ND50 /ml) was used. In some cases, neutralization activities were measured using a multiple-round viral infectivity assay using NP-2 cell lines [44]. Equal infectious titers of the viruses (100 tissue culture infectious dose) were incubated with serially diluted plasma samples (1/10 to 1/103) for 60 min at 37°C and used to infect the CD4+CXCR4+ NP-2 cells and CD4+CCR5+ NP-2 cells. After 60 min, the cells were washed once with phosphate-buffered saline. Culture supernatants were collected at 5 days after infection, and amounts of HIV-1 Gag p24 proteins were measured with a commercially available kit (RETROtek HIV-1 p24 Antigen ELISA, ZeptoMetrix Corporation, USA). Each plasma dilution was tested in duplicate, and the means of the p24 amounts were used to calculate the ND50.HIV-1 coreceptor usagesPrevious data of the coreceptor tropisms of the recombinant viruses [13], [30], [33] were used.MD simulationGp120 outer domain structures bearing various V3 elements were constructed with the homology modelling technique, using the Molecular Operating Environment, MOE 2006.08 (Chemical Computing Group Inc., Montreal, Quebec, Canada) as described [45], [46]. As the modelling template, we used the crystal structure of HIV-1 gp120 containing an entire V3 element at a resolution of 3.30Å (PDB code: 2B4C), which represents the structure after the CD4 binding [3]. The 251amino-terminal and 24 carboxyl-terminal residues were deleted to construct the gp120 outer domain structure. MD simulations were performed using the SANDER module in the AMBER 8 program package [47] with MDGRAPE-3 (http://mdgrape.gsc.riken.jp/) and the AMBER parm99 force field with the TIP3P water model. After heating calculations for 20 picoseconds until 310 K using the NVT ensemble, the simulations were executed using the NPT ensemble at 1 atm and at 310 K for 5 nanoseconds. Superimpositions of the structures were done by coordinating atoms of amino acids along the β-sheet at the V3 base.Supporting InformationFigure S1Information on the V3 sequences for the diversity analyses. Shown are the % distributions of CRF01_AE V3 sequences used in the present study (n = 1361) as a function of sampling years (a), countries (b), and V3 structural group (c). The sequences during 1991 to 2005 (n = 1148) represent a majority. They are mostly from Asia (15 countries, 1219 sequences). Others are from Africa (7 countries, 52 sequences), Europe (10 countries, 47 sequences), other regions (5 countries, 36 sequences), and unknown (1 sequence). V3 groups having CCR5 tropism (2b, 3b and 4b) represent the majority independent of the sampling period.(0.34 MB TIF)Click here for additional data file.Figure S2V3 diversity of HIV-1 subtypes A, B, and C. Global distribution (left) and dn/ds ratios (right) of V3 structural variants of HIV-1 subtypes A, B, and C were examined, using the HIV-1 public database information from June 2007, and plotted as described in Fig. 1C.(0.31 MB TIF)Click here for additional data file.Figure S3V3 amino acid sequences of the recombinant viruses. V3 sequences of the recombinant viruses are from CRF01_AE clones in uncultured peripheral blood mononuclear cells from a Japanese family [30] (V3 IDs of A1{similar, tilde operator }A9 and B1{similar, tilde operator }B13), A1 variants having naturally occurring basic amino acid substitutions (mt1{similar, tilde operator }mt8) [13], and TH09 isolate having the CRF01_AE consensus V3 sequence [33] (TH09). Deduced amino acids of the V3 sequences were aligned with the CRF01_AE consensus sequence, ENSI-c. The small blue open box indicates a potential N-linked glycosylation site conserved in the V3 structural group b. Red letters indicates basic amino acid substitutions with respect to ENSI-c. The large black box indicates 19 amino acid sequences used for V3-peptide ELISA in Fig. 2. The net charge is the number of positively charged amino acids (R, K, and H) minus the number of negatively charged amino acids (D and E). Coreceptor tropism of the recombinant viruses was determined using CD4+CXCR4+ HOS cells and CD4+CCR5+ HOS cells [13], [30], [33].(0.39 MB TIF)Click here for additional data file.Figure S4Effects of protein G on plasma neutralizing activities. The plasma samples (YM17 and YM61) were incubated with serially diluted protein G agarose solution (GammaBind Plus Sepharose, Amersham) for 60 min at 37°C. The agarose was removed by brief centrifugation, and the supernatants were used to measure ND50 against LAI recombinant viruses having non-rV3 (B1 and B10) using CD4+CXCR4+CCR5+ HeLa cells (MAGIC-5 cells [34]) as described in Materials and Methods.(0.21 MB TIF)Click here for additional data file.Figure S5Antibody epitope mapping of the rV3. Peptide-based, enzyme-linked immunosorbent assay [35] was carried out using indicated synthetic peptides matching the rV3 amino acids of the recombinant viruses (SI Fig. 3, recombinant ID of A1). Antibodies bound to the peptides were detected with anti-human IgG peroxidase conjugate and 3,3′,5,5′-tetramethylbenzidine substrate. Absorbance at 450 nm is shown.(0.27 MB TIF)Click here for additional data file.Figure S6ND50 in the single- and multiple-round viral infectivity assays. Plasma samples (n = 8) were used to measure ND50 against LAI recombinant viruses having rV3 (clone IDs of ENSI-c and A1) and non-rV3 (B6). The ND50 were measured in a single-round viral infectivity assay using CD4+CXCR4+CCR5+ HeLa cells (MAGIC-5 cells) [34] or a multiple-round viral infectivity assay using CD4+CXCR4+ NP2 cells and CD4+CCR5+ NP2 cells (NP-2 cells) [44] as described in Materials and Methods. Red diamonds indicate the medians of the neutralization titers of the 8 plasma samples.(0.20 MB TIF)Click here for additional data file.Figure S7Close-up view of the V3 base-stem region of Gp120 with non-rV3. 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+{
+    "id": "PMC2527524",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2527524\nAUTHORS: Jan Van den Stock, Wim A. C. van de Riet, Ruthger Righart, Beatrice de Gelder\n\nABSTRACT:\nMany people experience transient difficulties in recognizing faces but only a small number of them cannot recognize their family members when meeting them unexpectedly. Such face blindness is associated with serious problems in everyday life. A better understanding of the neuro-functional basis of impaired face recognition may be achieved by a careful comparison with an equally unique object category and by a adding a more realistic setting involving neutral faces as well facial expressions. We used event-related functional magnetic resonance imaging (fMRI) to investigate the neuro-functional basis of perceiving faces and bodies in three developmental prosopagnosics (DP) and matched healthy controls. Our approach involved materials consisting of neutral faces and bodies as well as faces and bodies expressing fear or happiness. The first main result is that the presence of emotional information has a different effect in the patient vs. the control group in the fusiform face area (FFA). Neutral faces trigger lower activation in the DP group, compared to the control group, while activation for facial expressions is the same in both groups. The second main result is that compared to controls, DPs have increased activation for bodies in the inferior occipital gyrus (IOG) and for neutral faces in the extrastriate body area (EBA), indicating that body and face sensitive processes are less categorically segregated in DP. Taken together our study shows the importance of using naturalistic emotional stimuli for a better understanding of developmental face deficits.\n\nBODY:\nIntroductionRecognizing faces of family and friends usually proceeds effortlessly. Yet a minority of people has difficulties telling apart who they are meeting with or remembering who they met previously when they can only go by the visual memory of the face. These problems can be quite dramatic, even to the point where they fail to recognize the face of their own spouse or child or for that matter their own face. The original reports of face recognition deficits for which the term prosopagnosia [1] was coined concerned cases of brain damage sustained in adulthood. More recently there have been reports of face recognition deficits that do not appear to be associated with any known neurological history. Although there are still only a few systematic reports of this condition, many more cases are described now compared to a decade ago and some authors have argued that as much as 2% of the population suffers from face recognition difficulties [2]. In analogy with developmental dyslexia these cases are now commonly referred to as developmental prosopagnosia (DP), referring to the possible origin of the adult face recognition deficit in anomalous development of the full face recognition skills. This behavioral deficit may include an anomaly in the putative congenital basis involved in the acquisition of the skill, but so far very little is known about this genetic basis and its importance for explaining behavioral deficits [3].Recent research on behavioral face recognition deficits and their neural basis has followed the leads from the reports on the neural basis of face recognition in normals as mainly revealed in fMRI studies over the last decade. There is now a consensus in the literature that face recognition is implemented in a network of brain areas [4], [5]. Among these, an area in the fusiform gyrus (FG), labeled the fusiform face area (FFA) [6], [7], has attracted most attention. Next to this area, the role of the inferior occipital gyrus (IOG) is repeatedly stressed in normal e.g. [8]–[10] and anomalous face recognition [11]. But it is fair to say that the functional significance of these two main areas for person recognition and its deficits is not yet entirely clear.Investigations of the neuro-functional correlates of DP with fMRI have yielded inconsistent results [11]–[16] (see Table 1 for an overview). The first fMRI-study including a DP case by Hadjikhani and de Gelder [11] found no face-specific activation in these two areas. A similar pattern was observed with another DP case [15]. On the other hand, other studies reported normal face-specific activation in developmental prosopagnosics (DPs) despite their severe behavioral deficits in face recognition [12]–[14], [16]. These findings suggest that intact functioning of the FFA and IOG are necessary, but not sufficient for successful face recognition.10.1371/journal.pone.0003195.t001Table 1Results from fMRI-studies on prosopagnosia.Studies with developmental prosopagnosicsNLesion localisationtaskcomparisonresultFFAIOGHadjikhani & de Gelder (2002)1n.a.Passive viewingFaces>objects−−Faces>houses+−Hasson et al. (2003)1n.a.One backFaces>buildings++Avidan et al. (2005)4n.a.One backFaces>(buildings & objects)++Bentin et al. (2007)1n.a.OddballFaces>places−−Faces>objects���−Degutis et al. (2007)1\n1n.a.One backFaces>scenes++Williams et al. 20071n.a.One backFaces>scenes+Van den Stock et al.3n.a.Oddball(Emotional+neutral faces)>housesdecreased neutral face activity+Studies with acquired prosopagnosicsNLesion localisationtaskcomparisonresultFFAIOGMarotta et al. (2001)2SM: right temporaloddballFaces>objectsSM(+)CR: right temporalCR−Hadjikhani & de Gelder (2002)2No evident lesionsPassive viewingFaces>objects−−GA: Faces>scramble−−RP: Faces>houses−+Rossion et al. (2003)1Right posterior inferior occipital to posterior fusiform gyrus; left middle fusiform gyrusOne backFaces>objects+Steeves et al. (2006)1Bilateral lateral occipital; left superior parieto-occipital sulcusOddballFaces>scenes+Sorger et al. (2007)1Right ventral occipito-temporal; right middle temporal gyrus; left FG, IOG and lingual gyrus; left medial cerebellum (see also Rossion et al. 2003)One backFaces>objects+(l)1This study reports about a training program administered to a patient. We report the fMRI result preceding the training.Abbreviations: n.a.: not applicable; FFA: Fusiform Face Area; FG: Fusiform Gyrus; IOG: Inferior Occipital Gyrus; SM, CR, GA & RP refer to subjects; +: significant activation; −: no significant activation; (l−): only left activation is observed.In view of the many different kinds of information a face provides (gender, age, emotion, familiarity, attractiveness etc.) and the different ways in which this information is called upon and used in daily life (whether the context only requires rapid detection that there is a face present, or on the contrary, full recognition of all facial attributes including name retrieval), it is worth stressing that the contextual requirements and the task settings are very important for evaluating face recognition problems and for understanding its neuro-functional basis and possible deficits. A finely tuned comparison of face recognition skills with other object recognition skills at the behavioral and neuro-functional level requires comparable task settings whether the object categories to be matched are faces or any other category that is suitable [17]–[21]. Since faces convey many different kinds of information it has so far been a daunting task to find a matching category to use as control stimuli. Previous approaches to find the best matching category have tended to explore either the physical similarity dimension (for example, using a continuum of more or less face like stimuli), the perceptual one or the functional one (for example, expertise with one or another specific object category). This has fed an ongoing debate about whether face processing mechanisms are qualitatively different from the processing mechanisms for objects (modularity hypothesis) [22], or on the other hand whether relative face specificity reflects the level of perceptual expertise with the stimulus category (expertise hypothesis) [9], [23]. As a matter of fact there are very few objects other than faces for which strong claims about category specific representation have been made. One exception concerns houses. Several studies report that this object category differentially activates a region around the collateral sulcus [24]–[26].An interesting object category not used so far concerns human bodies. Recently, it has been shown in normal subjects that perceiving human bodies or body parts activates an area in extrastriate cortex, labeled extrastriate body area (EBA) [27]. More recently a second body specific area was defined in the FG [28], [29]. This body sensitive area in FG overlaps at least partially with the face-sensitive one and it has been termed the fusiform body area (FBA). In parallel, recent findings show that the close similarities between face and body perception exist at the level of perceptual mechanisms as revealed by the inversion effect (a decline in performance for inverted stimuli compared to upright stimuli that is more pronounced for faces than for other object categories [30]), since the same inversion effect has been reported for bodies [31], [32] for reviews, see [33], [34].These behavioral and neuro-functional similarities between perceiving faces and bodies in normals and the fact that bodies represent a distinct but yet very closely related object category, raise the issue how bodies are processed in DP. A study by Duchaine et al. [35] presented natural faces and computer generated neutral body postures for testing face and body identity recognition in a DP patient using a sequential identity matching paradigm involving a minimal memory component. The performance of the patient was impaired for the faces, but within normal range for the bodies suggesting dissociation between face and body processing mechanisms with these task settings. Another study used event-related potentials (ERP) to investigate face and body perception in four DPs and found abnormal brain activation in the early time windows of the EEG (around 170 ms) for both faces and bodies in three of the four DPs [36].A second main objective of the present study is to investigate how the neural underpinnings of face and body processing in prosopagnosia are influenced by emotional information in the face and the body. As a matter of fact, the face-sensitive area in FG is well known from investigations of face recognition using neutral faces but it also figures predominantly in research on the neural basis of recognizing facial expressions. The presence of an emotion expression adds realism to the face but may also be an interesting developmental factor. Studies with younger subjects have predominantly reported higher activation for fearful faces, compared to neutral faces [37]–[41], but a recent study with both adolescents and adults found a reverse pattern in the FFA, namely higher activation for neutral than for fearful faces [42]. The mechanism of this emotional modulation in the FFA may be based on feedback loops with the amygdala [39], [41]. A similar explanation has been proposed for the increased activation in FG sensitive to body images representing an emotional expression [28].So far, the evidence concerning the neural correlates of processing emotional faces in DP is scarce. One study by de Gelder et al. [43] investigated this issue in acquired prosopagnosics (prosopagnosia occurring after brain damage). The included patients had lesions in either the FG, IOG or both. The results showed that the patients more strongly activated other face sensitive areas like the superior temporal sulcus (STS) or amygdala when they perceive facial expressions compared to neutral faces. The patients were also more accurate and faster in processing emotional faces compared to neutral faces, a finding that has been reported previously [44]–[46]. Since the patients in de Gelder et al. [43] had lesions in the ventral occipito-temporal cortex, the question arises how these brain areas respond to emotional information in prosopagnosics with severe face recognition problems but no known brain anomalies. To investigate this issue we presented the participants with neutral, fearful and happy facial and bodily expressions.MethodsParticipantsThe DPs were recruited after they had contacted us via our website or through reports in the popular press. All participants report life-long problems in recognizing people and typically complain about difficulties when meeting familiar persons unexpectedly and the ensuing social problems. AM (female) is a 54-year old housewife. She reports problems in recognizing others when meeting them outside the usual context, for example when she meets her parents in the supermarket. HV (male) is 43 years old and teaches writing and coaches in communication training. He experiences severe face recognition problems for as long as he can remember. LW (male) is a 48-year old university professor with longstanding difficulties for example in recognizing colleagues at conferences and students. None of the DPs had a neurological history and their structural MR-scans showed no abnormalities as judged independently by four experienced neurologists. The group of four control subjects was matched with the DP group on age, sex and educational level. All participants gave written informed consent according to the Declaration of Helsinki and the study was approved by the local ethics committee (CMO region Arnhem-Nijmegen, The Netherlands).Neuropsychological testingAll participants were presented with an extensive face recognition battery. Visual object recognition and face recognition were assessed with standard clinical tests and additional face and object perception experiments were run in sessions preceding the fMRI measurements. The neuropsychological tests and normative data are described elsewhere [36]. Face matching and face memory were tested with the Benton Face Recognition Test (BFRT) [47] and the Warrington Face Memory Test (WFMT) [48]. We used a computerized version of the latter test to obtain information about speed-accuracy trade-off. Basic visual functions were measured with the Birmingham Object Recognition Battery (BORB) (line length, size, orientation, gap, minimal feature match, foreshortened views and object decision) [49]. To investigate in detail different aspects of face perception, all participants were administered additional face and object perception experiments which have proven useful in previous investigations of face recognition and provided insight in processing strategies in prosopagnosia [4], [17], [36], [43], 50, 51.Like in our previous studies on prosopagnosia, the behavioral pattern of a normal inversion effects for faces compared to another single object category was measured with the faces and shoes task [17]. Participants were required to select the probe that corresponded with the identity of a simultaneously presented target. The target was always a frontal picture and the two probes underneath consisted of pictures in three quarter profile. Faces and shoes were presented upright and inverted for details, see [17], [50]. Feature-based processing was tested with a part-to-whole matching task which required participants to select the face-part probe (i.e., mouth or eyes) that was the same as that in the simultaneously presented whole face. The same procedure was followed for house-part probes (i.e., door or upper window) that had to be matched to the corresponding part in a whole house stimulus. Faces and houses were presented once upright and once inverted [4], [43]. Participants were instructed to respond as accurately and rapidly as possible. Accuracy and mean response-times were calculated for each test. We compared the accuracy and response times from the upright stimuli with the inverted stimuli in one-tailed paired-sample t-tests. A significantly lower accuracy or longer response time for the inverted stimuli is defined as an inversion effect, whereas a higher accuracy or shorter response time for the inverted stimuli is defined as a paradoxical inversion effect. Data of the control group were normalized and z-scores were obtained for every DP.fMRI measurementsStimulus materialsThe face and body stimuli were used previously in an fMRI investigation of the neural substrates of processing face and body perception in neurologically intact observers [52]. Pictures of fearful, happy and neutral faces were taken from the Karolinska Directed Emotional Face database [53]. From our own database, pictures of fearful and happy bodily expressions, instrumental (emotionally neutral) bodily expressions (pouring water into a glass) and houses were used. We used houses as stimuli for the control condition, because they constitute a single object category that has been extensively explored in other studies and is known to elicit activation in specific brain areas [24]–[26]. Instrumental body expressions were used because, like emotional expressions, these displays elicit action representation and implicit movement [54], and hence constitute a balanced comparison category for the emotional expressions. All images of faces and bodies were previously validated regarding emotional expression (minimum recognition rate: 75%). For further details concerning the validation procedure, see [52].A total of 42 images was used, six in every condition (fearful faces, happy faces, neutral faces, fearful bodies, happy bodies, neutral bodies and houses). There was no identity overlap between faces and bodies or between the emotions. Faces were fitted inside a gray oval shape, which masked external aspects of the faces. Body and house stimuli were cut out, removing all background. The faces of the body stimuli were covered with a gray opaque mask. Additionally, one picture of a chair was used as an oddball stimulus. All stimuli were resized to 300 pixels in height and presented on a gray background.ProcedureThe design was adapted from our previous study [52]. In order not to exacerbate the face handicap of the DP group, we modified the experimental paradigm from a facial expression categorization task to an oddball detection task thereby also avoiding selective attention to the faces with an emotional expression. Moreover, this procedure excludes that activation profiles are contaminated by motor responses in the conditions of interest while still providing control data on attention to the stimuli. A trial started with the presentation of a fixation cross (200 ms), followed by a stimulus (500 ms) and finally by a gray screen (2200 ms) (see Figure 1). All stimuli were presented six times in random order in an oddball paradigm (participants were instructed to press a response button when a chair was shown). The session consisted of 288 trials (7 conditions×6 identities×6 presentations, plus 36 oddball trials). Additionally, 96 null-events consisting of a gray screen lasting the whole trial length were included to reduce stimulus onset predictability and to establish a baseline [55]. The experiment was preceded by a short practice-session which used a different set of face and body stimuli.10.1371/journal.pone.0003195.g001Figure 1Schematic representation of the experimental design.Participants were instructed to press the response button when a chair was presented.Participants lay supine in the scanner with head movements minimized by an adjustable padded head holder. Stimuli were projected onto a mirror above the participant's head. Responses were recorded via an MR-compatible keypad (MRI Devices, Waukesha, WI), positioned on the right side of the participant's abdomen. A PC running Presentation 9.70 (Neurobehavioral Systems, San Francisco, CA) controlled stimulus presentation and response registration.Image AcquisitionImages were acquired using a 1.5 Tesla Sonata scanner (Siemens, Erlangen, Germany). Blood oxygenation level depend (BOLD) sensitive functional images were acquired using a single shot gradient echo-planar imaging (EPI) sequence [TR (repetition time) = 3790 ms, TE (echo time) = 40 ms, 43 transversal slices, ascending acquisition, 2.5 mm slice thickness, with 0.25 mm gap, FP (flip angle) = 90°, FOV (field of view) = 32 cm]. An automatic shimming procedure was performed before each scanning session. A total of 312 functional volumes were collected for each participant. Following the experimental session, structural images were acquired using an MP-RAGE sequence [TR/TE/TI (inversion time) 2250 ms/3.93 ms/850 ms, voxel size 1×1×1 mm].ResultsNeuropsychological testingAll DPs scored outside the normal range for the BFRT and/or the WFMT, but none showed an anomalous score on more than one subtest of the BORB suggesting that the visual recognition difficulties of the DPs as measured by these two clinical tests are not due to basic visual perception problems diagnosed in the BORB (see Table 2). AM scored significantly below the mean on the BFRT and WFMT, for both accuracies and response times. HV had a borderline performance on the BFRT and prolonged response times on the WFMT. LW scored within normal range on the BFRT, but on the WFMT both accuracy and response times were anomalous.10.1371/journal.pone.0003195.t002Table 2Results from neuropsychological testing.ControlsAMHVLWBFRT accuracy (/54)45.4 (A)28 (SI)40 (BL)44 (A)WFM accuracy (/50)44.029***4134**WFM RT (ms)17783171***3853 ***3171 ***Faces accuracy (/64)Upr63.357***6364Inv62.056**6257**Shoes accuracy (/64)Upr62.5646461Inv62.8626458**Faces RT (ms)Upr11463743 ***2840 ***1741**Inv15263406 ***3640 ***2112Shoes RT (ms)Upr9782533 ***1757 ***1450***Inv10692849 ***1689 ***1673**Face-parts accuracy (/64)Upr62.747***60*59**Inv62.052***6356**House-parts accuracy (/64)Upr62.7636261Inv63.2646463Faceparts RT (ms)Upr15622099**4446 ***3462***inv175522244130 ***3229***Houseparts RT (ms)Upr11921554*1703 ***1917***inv113213611593 ***1774***Response times are shown for correct responses. Comparisons of DP's and matched controls are made by z-scores on the basis of the following control groups:Control group for the Warrington face memory: N = 25 (18–27 yrs).Control group for the Faces and Shoes task: N = 11 (18–28 yrs).Control group for the Face- and Houseparts: N = 21 (18–29 yrs).Asterisks indicate P-values corresponding to the Z-scores. * p<.05; ** p<.01; *** p<.001. SI: severe impairment; BL: borderline; A: average.To measure face and object recognition in a comparable way and assess relative configural processing routines, we compared upright and inverted stimulus matching for each object category [17], [36]. The control group showed an inversion effect for matching faces in both the accuracy (t(10) = 1.892, p<.05) and response time (t(10) = 3.164, p<.005). The controls showed no inversion effect for matching shoes. For the DPs, the response times were high as previously reported [4], [36]. AM was impaired in matching both upright (Z<−5.75) and inverted (Z<−3.39) faces. Her response times showed a paradoxical inversion effect pattern for matching faces and a normal inversion for matching shoes. HV had accuracies within the normal range, but displayed a normal inversion pattern in the response times for matching faces and a paradoxical inversion effect in the response times for matching shoes. LW showed reduced accuracy for matching inverted faces (Z<−2.82) and inverted shoes (Z<−2.74). His response times for matching upright faces were prolonged (Z>2.39), while the latencies for inverted faces were on average. He displayed the normal inversion pattern for matching faces and shoes in both accuracy and response times.Feature-based matching was tested with the faces and houses task see [4] for details. The control group showed a normal inversion effect for matching face parts in accuracy (t(10) = 1.746, p<.05) and in response time (t(10) = 4.754, p<.001). However, they showed a paradoxical inversion effect for matching house-parts in accuracy (t(10) = 1.743, p<.05) and response time (t(10) = 2.667, p<.01). AM showed lower accuracies for matching both upright (Z = −11.81) and inverted (Z = −5.36) face-parts. Her latencies for matching upright face-parts (Z = 2.51) and house-parts (Z = 2.06) were higher than normal. She displayed a paradoxical inversion effect in the accuracy data for matching face-parts and house parts, and in the response times for matching house-parts. Her response times for matching face-parts showed a normal inversion pattern. HV had a reduced accuracy for matching upright face-parts (Z = −2.00). He also had highly prolonged response times for upright faces (Z = 13.46) and to a lesser extend for inverted faces (Z = 8.27). Latencies for upright houses (Z = 2.28) and inverted houses (Z = 3.31) were also prolonged, but less than for faces. HV showed paradoxical inversion effects in both the accuracy and response times for face-part and house-part matching. LW's accuracy for matching upright (Z = −2.76) and inverted (Z = −3.16) faces was impaired. His responses for matching upright face-parts (Z = 8.87), inverted face-parts (Z = 5.13), upright house-parts (Z = 4.12) and inverted house-parts (Z = 4.61) were prolonged. LW's accuracy data showed a normal inversion pattern for matching face-parts and a paradoxical inversion pattern for matching house parts. He displayed a paradoxical inversion effect in his response times for matching face-parts and house-parts.fMRI analysisAll participants performed flawlessly on the oddball detection task.PreprocessingImaging data were analyzed using Brainvoyager QX (Brain Innovation, Maastricht, the Netherlands). The first five volumes of each functional run were discarded to allow for T1 equilibration. Pre-processing of the functional data included 3D-motion correction, slice scan time correction, temporal data smoothing (high pass filter 3 cycles in time course) and spatial smoothing with an isotropic 6-mm full-width-half-maximum (FWHM) Gaussian kernel. Images were spatially normalized to Talairach space [56] and resampled to a voxel size of 1×1×1 mm. Statistical analysis was based on the general linear model (GLM), with each condition defined as a separate predictor. Null-events were modeled explicitly.ROI definitionWe used a “split-half” method for defining regions of interest (ROI), in order to be sure that the observed effects are not due to a selection bias [57]. The even trials were used to define the ROIs and the odd trials were used for the within ROI analysis. To localize face-sensitive activation in FG, i.e. FFA, we contrasted the even trials of all face conditions (fearful, happy and neutral) with houses (all trials) and identified significant voxels in each subject within a restricted region of the FG (Talairach y-coordinate between −25 and −65). The voxel set comprising this activation determined the ROI, in this case the FFA. The same procedure was followed in a restricted region of the IOG (Talairach y-coordinate <−70). To identify body sensitive areas, we compared the even trials of all bodies (fearful, happy and instrumental) with houses and mapped the selective activation in a restricted region of FG to determine the FBA (Talairach y-coordinate between −25 and −65) and the region around the junction of the middle temporal and middle occipital gyrus to determine the EBA (Talairach x-coordinate between 25 and 60; y-coordinate between −55 and −75; z-coordinate between −15 and 15). We used a liberal threshold (p<.05, uncorrected). Since previous studies reported that cortical face and body selective regions are often weaker or even absent in the left hemisphere [6], [29], we restricted the analysis to the right hemisphere.Smoothed activation maps are projected on the inflated right hemisphere of one subject. For every ROI, the activation maps of the control subjects are collapsed and the result is displayed by the black contours. This procedure allows visualization of the spatial extent of the activation across different subjects. Activation of the individual DPs is plotted in color (see Figures 2 to \n\n5). The Talairach coordinates of the activation maps are shown in Table 3.10.1371/journal.pone.0003195.g002Figure 2Face-specific activation in right FG when comparing faces (fearful/happy/neutral) with houses.Left: Areas are shown on an inflated right hemisphere. Activation maps of the control subjects are collapsed and displayed by the black contours. Activation of the individual DPs is plotted in color. Right: beta-values by condition, group and DP. Error bars represent one standard error of the mean (SEM). Conditions represent from left to right: fearful faces, happy faces, neutral faces, fearful bodies, happy bodies, neutral bodies and houses. White columns display the average value of the three patients. Black columns show the average value of the controls. Triangles represent the individual values of the DPs.10.1371/journal.pone.0003195.g003Figure 3Face-specific activation in right IOG when comparing faces (fearful/happy/neutral) with houses.Left: Areas are shown on an inflated right hemisphere. Activation maps of the control subjects are collapsed and displayed by the black contours. Activation of the individual DPs is plotted in color. Right: beta-values by condition, group and DP. Error bars represent one SEM. Conditions represent from left to right: fearful faces, happy faces, neutral faces, fearful bodies, happy bodies, neutral bodies and houses. White columns display the average value of the three patients. Black columns show the average value of the controls. Triangles represent the individual values of the DPs.10.1371/journal.pone.0003195.g004Figure 4Body-specific activation in right FG when comparing bodies (fearful/happy/instrumental) with houses.Left: Areas are shown on an inflated right hemisphere. Activation maps of the control subjects are collapsed and displayed by the black contours. Activation of the individual DPs is plotted in color. The purple indicates overlap between red (AM) and blue (LW). Right: beta-values by condition, group and DP. Error bars represent one SEM. Conditions represent from left to right: fearful faces, happy faces, neutral faces, fearful bodies, happy bodies, neutral bodies and houses. White columns display the average value of the three patients. Black columns show the average value of the controls. Triangles represent the individual values of the DPs.10.1371/journal.pone.0003195.g005Figure 5Body-specific activation in right EBA when comparing bodies (fearful/happy/instrumental) with houses.Left: Areas are shown on an inflated right hemisphere. Activation maps of the control subjects are collapsed and displayed by the black contours. Activation of the individual DPs is plotted in color. The purple indicates overlap between red (AM) and blue (LW). Right: beta-values by condition, group and DP. Error bars represent one SEM. Conditions represent from left to right: fearful faces, happy faces, neutral faces, fearful bodies, happy bodies, neutral bodies and houses. White columns display the average value of the three patients. Black columns show the average value of the controls. Triangles represent the individual values of the DPs.10.1371/journal.pone.0003195.t003Table 3Number of voxels (N) and Talairach coordinates (range) of ROIs.ContrastMean Controls (range)AMHVLWAreaNxyzNxyzNxyzNxyz(fF+hF+nF)>HFFA427 (9:2208)40 (31:48)−42 (−59:−27)−15 (−33:−5)2439−60−2044836−64−914041−34−16IOG742 (15:2667)32 (15:55)−81 (−95:−70)−7 (−21:9)924−82−1453425−74−1315647−71−10(fB+hB+iB)>HFBA1220 (303:2282)44 (27:58)−40 (−60:−25)−15 (−23:−19)40634−49−950241−40−183441−54−11EBA416 (42:1201)45 (33:59)−63 (−74:−52)4 (−11:15)73743−70−680236−66−10240443−688fF>nFRight AMG146(146:146)12(8:16)−8(−4:−12)−12(−14:−10)14240−820230−7Left AMG15−20−8−380−241−5hF>nFRight AMG11(0:11)9(8:9)−1(−2:0)−11(−12:−10)23230−8Left AMG265(0:265)−13(−18:−11)−12(−14:−9)−14(−18:−19)35−24−1−6fB>nBRight AMG258(8:779)19 (12:26)−7 (−9:0)−12 (−17:−7)14213−6−6Left AMG351 (0:710)−20 (−28:−11)−8 (−9:0)−10 (−19:18)130−23−1−18120−17−10−11hB>nBRight AMG152 (0:330)19 (13:25)−5 (−14:0)−16 (−21:−7)Left AMG659 (0:819)−17 (−24:−11)−4 (−11:0)−13 (−21:−7)102−19−11−6The coordinate range represents the outer voxels of the collapsed cluster from all controls. Abbreviations: n = neutral; f = fearful; h = happy; i = instrumental; F = face; B = body; H = house; FFA = Fusiform Face Area; IOG = Inferior Occipital Gyrus; FBA = Fusiform Body Area; EBA = Extrastriate Body Area; AMG = Amygdala.Effects of emotional contentThe analyses were performed on the beta-values of the odd trials of the conditions. To investigate differences between the DP group and the control group, we used independent samples t-tests, corrected for unequal variances (in degrees of freedom).FFA\nFigure 2 shows the smoothed face-specific activation (left) and the beta-values of all conditions (right) in FG. The controls show the expected age-dependend higher activation for neutral than for fearful expressions [42]. We calculated the difference between fearful faces and neutral faces and this difference was significantly larger in the control group (t(4.946) = −2.583, p<.05). The difference between happy faces and neutral faces was marginally significantly different between groups (t(4.906) = −2.051, p<.097). Since previous studies showed a lower activation for faces in DPs compared to controls [11], [15], we used one-tailed post-hoc t-tests to compare the activation levels of the three face conditions between both groups. This revealed a marginally significant difference for the neutral faces (t(4.980) = 1.929, p<.051).IOG\nFigure 3 shows the smoothed face-specific activation (left) in IOG and the beta-values of all conditions (right). A t-test on the difference between fearful faces and neutral faces showed no significant difference between both groups (t(4.510) = .0233, p<.826). The difference between happy faces and neutral faces was also not significantly different between the DPs and controls (t(4.989) = −1.235, p<.272).FBA\nFigure 4 shows the smoothed body-specific activation (left) and the beta-values of all conditions (right) in FBA.The difference between either fearful bodies (t(4.475) = −.088, p<.934) or happy bodies (t(4.567) = .321, p<.762) and instrumental bodies was not significantly different between both groups.EBA\nFigure 5 shows the smoothed body-specific activation (left) and the beta-values of all conditions (right) in EBA.The difference between fearful bodies and instrumental bodies was not different between groups (t(3.786) = 1.153, p<.317). A t- test on the difference between happy and instrumental bodies revealed no significant between-group difference (t(3.722) = .339, p<.573).Effects of categorical selectivityTo investigate the selectivity of processing faces and bodies in the brain, we calculated the difference between the mean of the three face conditions and the mean of the three body conditions in FFA and IOG. A comparison using t-tests showed that this difference was smaller in the control group in IOG, but it did not reach statistical significance (t(3.961) = 2.122, p<.102). We also calculated the difference between the mean of all body conditions and the mean of all face conditions in FBA and EBA. Independent sample t-tests showed no significant between-group differences.Processing of neutral facesSince the main body of research on DP concerns neutral faces, we compared the activation level of neutral faces between both groups in all four ROIs, using t-tests. In addition to the above mentioned difference in FFA, this revealed a marginally significantly higher activation for neutral faces in EBA in the DP group (t(4.955) = 2.044, p<.097).Effects of emotion in amygdalaFinally, we performed a post-hoc analysis, in which we defined the amygdala in each subject, based on the individual anatomy. This ROI consisted in each hemisphere of a cube of 13×13×13 voxels around the center of the amygdala and we performed a second GLM in this area. The results are shown in Table 3. Contrasting fearful faces with neutral faces revealed significant activation in all three patients (left amygdala in AM; bilateral amygdala in HV and right amygdala in LW). Comparing happy with neutral faces showed activation in two patients (left amygdala in HV and right amygdala in LW). Fearful compared with neutral bodies differentially activated the amygdala in two patients (left amygdala in AM and bilateral amygdala HV). Happy bodies triggered significantly more amygdala activity in one DP (left amygdala in HV) compared to neutral bodies.DiscussionThe first major finding is that compared to the control group, the DP group displays a similar activation level for the emotional faces, but a lower activation in FFA for neutral faces. A lower activation level in DP for neutral face perception in FG is consistent with earlier reports [11], [15]. The present results are compatible with the theoretical perspective on face recognition difficulties argued for previously [18], [21] suggesting a higher threshold for neutral face recognition performance in prosopagnosics. This relative difficulty with neutral faces is based on the notion that faces are more difficult stimuli than many other categories they are routinely compared with.Emotional stimuli trigger a higher level of arousal e.g. [58], [59] and emotion in a face constitutes an additional feature that carries important communicative information and is therefore more salient. This saliency hypothesis is supported by a number of behavioral studies, with different visual tasks, that have demonstrated that adding emotional information to a face results in a greater tendency to capture attention [60]–[63]. Note though that the emotion effects we observe are not specific for emotions with a negative valence since we obtain similar effects for both fearful and happy (although less pronounced) expressions.However, normal FFA activation for facial expressions in the presence of lower than normal activation for neutral faces suggests that the activation boost is triggered more in he emotion processig than in the impaired face processing system in ventro-temporal cortex. Studies on perception of emotional faces in normals have hypothesized the existence of a feedback mechanism between FG and amygdala [38], [39], [64]–[66]. The possibility that such feedback connections from the amygdala may be active in prosopagnosia and boost face processing was already suggested in an earlier study of emotional faces in prosopagnosia [43]. Two acquired prosopagnosics were presented with both a neutral and emotional part-to-whole face matching task. The patients had lesions in FG and/or IOG, but the results showed normal activation in other face-sensitive area's (amygdala, superior temporal sulcus), for the contrast between emotional and neutral faces. The patients were also more accurate and faster when they performed the task with emotional faces compared to neutral ones. Moreover, the patients showed a normal inversion effect for matching emotional but not for neutral faces.Lower neural activity in the DPs for neutral faces, but not for emotional faces is compatible with a dual route model of face perception as argued first in de Gelder and Rouw [4] and adapted in de Gelder et al. [43], involving subcortical structures along a pathway that is able to proces facial expressions (the pulvinar-superior colliculus-amygdala route) [67] which in turn may boost face representations in the cortical route in temporal cortex even when face representations in temporal cortex are weak as shown by the lower activation for neutral faces in the DP group [43]. The pattern observed here is in line with this and may also explain why emotional content facilitates the cortical processing of faces in prosopagnosia. Consistent with this, we observed a higher activity level of the amygdala for emotional faces compared to neutral ones. A related and more extreme phenomenon is observed in hemianopic patients, who are unable to consciously report the presentation of a face in the blind visual field and do not show FG activation when presented facial expressions in the blind field but who perform well above chance in tasks where they have to guess the facial expression [68].Our second main finding concerns the categorical specificity of face vs. body representation in DPs. We compared the activation of body conditions in the face selective regions and of the face conditions in the body selective regions between both groups. On the one hand, our findings indicate that perceiving neutral faces results in a higher activation of EBA in the DP group, compared to the control group. Combined with the lower activation for neutral faces in FFA, this increased activation in EBA might indicate an anomalous cerebral processing route in DP. It may be the case that (neutral) faces are processed in the areas more dominantly dedicated to body perception. On the other hand, we find a higher activation for perceiving bodies in IOG. These combined findings indicate that the neural correlates of perceiving faces and bodies, as manifested in IOG and EBA show a lower degree of specificity in DP.For body triggered activity we find no difference in neutral vs. emotional expressions between both groups, either in FBA or EBA. This indicates that the anomalous neuro-functional substrate in our DP group for neutral faces does not extent to the processing of bodies and bodily expressions. This is in line with recent behavioral data showing no impairment in recognizing neutral body postures in one DP patient [35]. One of the DPs (HV) in the present study participated in a previous ERP study on perception of neutral faces and neutral bodies [36] and the results of both studies are partly converging. Righart & de Gelder [36] measured the electrical brain correlates of the inversion effect as an index of configural processes (the ability to perceive stimuli as one configuration as opposed to an assemblage of features [69]). HV differed significantly from the control group in face processing on two accounts. He displayed a paradoxical ERP inversion effect (the reverse pattern from the controls) around 100 ms after stimulus presentation (P1 amplitude) and no inversion effect around 170 ms after stimulus presentation (N170 latency). But his results for bodies did not differ from the controls.An important and relevant difference between face and body perception concerns the coding of identity. A face contains all necessary information about the identity of a person and we are used and trained to recognize identity by the face. A person can be readily identified on the basis of his face, but identification based on the body alone is far less evident. The different pattern in FG for faces and bodies may therefore reflect the possibility that FG is more involved in processing person identity [7] which is typically more based on the face than on the body.Notwithstanding the well documented involvement of FG in face perception, its precise role of FG in prosopagnosia is still a matter of debate. We do not clearly understand at present how factors like maturation of different cortical areas, like the FG, are important for normal face recognition. Reduced volume of the right temporal lobe has previously been reported in a DP patient [70]. A structural imaging study in six DP subjects investigated volumetric and morphometric properties in occipito-temporal cortex and showed a decreased volume of the FG that correlated with face recognition deficits [71]. At the neuro-functional level, recent data collected from normals show a correlation between the volumetric size of the right FFA and recognition memory for neutral faces [72]. This study also investigated the development of category specific brain areas and the results suggest that the relative size of the FFA increases during development. Moreover, the development of the FFA takes longer compared to that of object selective areas (lateral occipital complex) or face sensitive areas in the superior temporal sulcus see [73] for review and discussion. These findings support the notion that DP may be associated with abnormal development of FG which may be either a consequence or a cause of anomalous face skills. Lesions in acquired prosopagnosia (AP) patients often include the FG e.g. [43], [74], although other cases have also been reported with lesions more posterior than the face sensitive part of the FG e.g. [75], [76]. Besides the heterogeneity across lesion localization in AP, considerable heterogeneity consists in behavioral symptoms in DP [77]. 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+{
+    "id": "PMC2527560",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2527560\nAUTHORS: Kiet A Ly, Christine A Riedy, Peter Milgrom, Marilynn Rothen, Marilyn C Roberts, Lingmei Zhou\n\nABSTRACT:\nBackgroundHabitual consumption of xylitol reduces mutans streptococci (MS) levels but the effect on Lactobacillus spp. is less clear. Reduction is dependent on daily dose and frequency of consumption. For xylitol to be successfully used in prevention programs to reduce MS and prevent caries, effective xylitol delivery methods must be identified. This study examines the response of MS, specifically S. mutans/sobrinus and Lactobacillus spp., levels to xylitol delivered via gummy bears at optimal exposures.MethodsChildren, first to fifth grade (n = 154), from two elementary schools in rural Washington State, USA, were randomized to xylitol 15.6 g/day (X16, n = 53) or 11.7 g/day (X12, n = 49), or maltitol 44.7 g/day (M45, n = 52). Gummy bear snacks were pre-packaged in unit-doses, labeled with ID numbers, and distributed three times/day during school hours. No snacks were sent home. Plaque was sampled at baseline and six weeks and cultured on modified Mitis Salivarius agar for S. mutans/sobrinus and Rogosa SL agar for Lactobacillus spp. enumeration.ResultsThere were no differences in S. mutans/sobrinus and Lactobacillus spp. levels in plaque between the groups at baseline. At six weeks, log10 S. mutans/sobrinus levels showed significant reductions for all groups (p = 0.0001): X16 = 1.13 (SD = 1.65); X12 = 0.89 (SD = 1.11); M45 = 0.91 (SD = 1.46). Reductions were not statistically different between groups. Results for Lactobacillus spp. were mixed. Group X16 and M45 showed 0.31 (SD = 2.35), and 0.52 (SD = 2.41) log10 reductions, respectively, while X12 showed a 0.11 (SD = 2.26) log10 increase. These changes were not significant. Post-study discussions with school staff indicated that it is feasible to implement an in-classroom gummy bear snack program. Parents are accepting and children willing to consume gummy bear snacks daily.ConclusionReductions in S. mutans/sobrinus levels were observed after six weeks of gummy bear snack consumption containing xylitol at 11.7 or 15.6 g/day or maltitol at 44.7 g/day divided in three exposures. Lactobacillus spp. levels were essentially unchanged in all groups. These results suggest that a xylitol gummy bear snack may be an alternative to xylitol chewing gum for dental caries prevention. Positive results with high dose maltitol limit the validity of xylitol findings. A larger clinical trial is needed to confirm the xylitol results.Trial registration[ISRCTN63160504]\n\nBODY:\nBackgroundMutans streptococci (MS), more specifically S. mutans and S. sobrinus, are implicated in the development of dental caries in humans [1]. Xylitol, a naturally occurring sugar alcohol approved for use in food by the U.S. Food and Drug Administration (FDA) since 1963, has been shown to reduce MS levels in plaque and saliva and to markedly reduce tooth decay. Studies involving schoolchildren demonstrated that habitual use decreased dental caries.A recent review summarized the availability of xylitol-containing products on the internet and in supermarkets and other commercial outlets in the U.S. and included an assessment of their potential to provide a minimally effective dose (6 g) to reduce MS and tooth decay [2]. The review reports that, aside from chewing gum and lozenges, there have been few clinical studies performed on other xylitol-containing products. In all, the existing studies suggested that xylitol can reduce MS levels in saliva and plaque and reduce tooth decay. However, these prospective trials were not designed to assess the relationship between dose or frequency of xylitol use and reductions in MS level or tooth decay. To fill this gap, our group conducted studies to determine the minimal effective dose [3] and frequency [4] of xylitol use when delivered via chewing gum. These studies concluded that xylitol chewing gum dose of 6.9 to 10.3 g divided into at least three uses per day is efficacious in reducing MS, specifically S. mutans and S. sobrinus (herein referred to as S. mutans/sobrinus), level in plaque and saliva. On the other hand, 3.4 g/day or frequencies of use less than 3 times/day were not statistically different from controls even though small reductions were observed.Xylitol chewing gum and lozenges are widely available and used by consumers in Europe and in Korea, Japan, Thailand, and China. Finland was the first country to implement a national campaign, \"Smart Habits\" xylitol, to promote xylitol use to reduce tooth decay in children [5]. Similar promotions are occurring in other European and Asian countries especially Japan and Korea where xylitol chewing gum has captured nearly 50% of the chewing gum market. More recently, the U.S. Army implemented the \"Look for Xylitol First\" initiative to promote xylitol use among deployed troops to improve their oral health [6] and have begun to include xylitol chewing gum in \"meals ready to eat\" (MRE) rations. Similar programs to address tooth decay in U.S. children have not been adopted in part because chewing gum and hard candies consumption are considered choking hazards and thus not acceptable xylitol delivery vehicles for children [7]. For xylitol to be successfully used in oral health prevention programs for U.S. children, effective means of delivering xylitol in a therapeutic dose and frequency must be identified.The purpose of this RCT was to test the hypothesis that six weeks of habitual consumption of a xylitol gummy bear snack is effective in reducing S. mutans/sobrinus in plaque and to lay the groundwork for a dental caries trial. This study also evaluated the effect of xylitol on Lactobacillus spp.MethodsSubjectsSubjects (n = 154) were first to fifth grade children attending Morton and White Pass elementary schools in rural Washington State, USA. The children were initially presented a skit with an oral health message during a general assembly to introduce them to the University of Washington research team. A partnership between the research team and each school was established. The school sent parents an informational letter describing the study and asked permission for their child's participation in the \"Gummy Bear Study\" (see Additional file 1). Parents interested in having their children participate signed and returned the enclosed consent form along with a brief general health questionnaire (see Additional file 2). Children with reported antibiotic use during the previous two weeks or anticipated its use during the study period were excluded, as were children with a history of gastrointestinal problems. At the first study visit, the assent of each child was obtained prior to study procedures. The University of Washington Institutional Review Board approved the study and related materials.Study designThis prospective double-blind, randomized trial employed a three-group design. Children received either 15.6 g (X16) or 11.7 g (X12) xylitol/day, or maltitol (M45) 44.7 g/day. Maltitol was used as the null-comparison because it is only slowly fermentable and a previous study reported no adaptation by MS in plaque to maltitol or xylitol compared to sorbitol and sucrose [8]. Because the gummy bears might be hastily chewed, resulting in small chunks which then would be swallowed thus reducing the xylitol oral bioavailability, higher xylitol doses were used than in previous gum or lozenges studies.The study design controlled for the frequency and number of gummy bears consumed. Dose of xylitol was varied by combining xylitol and maltitol gummy bears. Pre-packaged and labeled gummy bear unit-doses were distributed in the classroom during school hours three times per day. Gummy bears were not sent home on non-school or missed school days. Subjects were randomly assigned to groups using a computer generated block randomization procedure to ensure a similar proportion of participants in each group. The group assignments were kept by the biostatistician and were decoded at the end of the study. Study and school staff and subjects were blind to group assignment.Sample sizeThe sample size was determined based on intent-to-treat and to provide sufficient power to test the hypothesis that xylitol gummy bears would reduce S. mutans/sobrinus and Lactobacillus spp. levels in plaque by ≥ 0.75 log10 after 6 weeks of exposure. A sample size of n = 41 subjects per group provided 80% power (2-sided, α = 0.05), where a difference in total bacteria counts between pre- and post-intervention was assumed to be 0.75 log10 = 5.6-fold reduction. Assuming a loss to follow-up of 10% to 20%, a total recruitment of 51 subjects per group (total n = 153) was necessary to ensure adequate minimum sample size.Gummy bears & unit-dose packagingBoth the xylitol and maltitol gummy bear snacks used in this study were produced by Santa Cruz Nutritionals (Santa Cruz, CA) especially for this study and are not available in the general market. The gummy bear snacks looked identical having the same weight (5 g/gummy bear), size, colors (red and green) and flavor (strawberry). They had similar texture and sweetness. Each gummy bear was 30 × 20 × 15 mm in size (see Figure 1). Danisco (Redhill, UK) and Santa Cruz Nutritionals (Santa Cruz, CA, USA) provided the proprietary formulations for the xylitol and maltitol gummy bears, respectively. Each 5 g xylitol gummy bear piece contained 1.3 g (26%) xylitol, 2.7 g (54%) polydextrose, water and gelatin, and minuscule amounts (<1.5%) of citric and lactic acid, mineral oil, carnauba wax and sucralose. Each maltitol gummy bear contained 3.7 g (74%) maltitol, water, gelatin, and similar minuscule amounts of the other ingredients found in xylitol gummy bears. Both types of gummy bears contained similar amounts of substances with laxative effects, i.e. maltitol gummy bear = 3.7 g, xylitol gummy bear = 4.0 g (1.3 g xylitol + 2.69 g polydextrose). However, polydextrose, a non-digestible polysaccharide, has a lower threshold (~90 g/day) for side effects [9] thus the laxative effects may have been slightly higher for the xylitol gummy bear groups.Figure 1Photograph of Xylitol and Maltitol Gummy Bears.Study personnel pre-packaged the gummy bears in unit-doses for classroom distribution three times a day: 15.6 g/day = four xylitol gummy bears; 11.7 g/day = three xylitol and one maltitol gummy bears; and maltitol control = four maltitol gummy bears per unit-dose for a total of 12 gummy bears per day for all three groups. Unit-dose packages were labeled with the appropriate randomized ID numbers, date, and period of distribution to facilitate tracking of the administrations each child received per day.Gummy bears distribution and adherence proceduresUnit-dose packages were ordered by day and session for each child and pre-packed into bins by classroom before being delivered to the schools weekly. School principals identified people who regularly served as classroom volunteers to hire as community workers for the study. Those women received training in the Responsible Conduct of Research at the University of Washington and were trained to distribute and monitor consumption of the gummy bear snacks in the classroom. The community workers established a schedule with the teachers that allowed them to cover all classrooms three times over the course of the school day at approximately even intervals. The unit-dose packages were distributed according to the child's assigned ID number, date and session number. The community workers observed the consumption of the gummy bears and immediately collected and recorded any unconsumed gummy bears, including those for any absent children. Classroom logs were faxed to the research staff daily to monitor study progress. Incompletely consumed packages of gummy bears were stored in the staff workroom and were picked up by the research staff weekly and checked against the classroom logs for accuracy.To increase acceptance of the gummy bear snacks and to permit adaptation of the gastro-intestinal system to the polyols, the first week of the study was designed as a ramp-up period. All groups consumed one unit-dose (four gummy bears) corresponding to their group assignment (X16, X12, or M45) on day one and gradually increased to the full three unit-doses (12 gummy bears) by day six. Parents were provided with information on and encouraged to report to the community workers laxative effects common to polyol consumption such as bloating, stomach cramps, flatulence, loose stool, or diarrhea that their child might experience. During the third week of the study, parents were sent a questionnaire on their child's side effects experiences.Plaque sampling and cultureTrained research staff collected plaque samples from the children at enrollment (baseline) and six weeks later. Research staff collected samples on-site in the stage area of the assembly hall, from one to four classrooms, throughout the school day. No special instructions were given regarding tooth brushing on collection day. As with our previous xylitol dose and frequency studies, research staff collected samples from the cervical third of the buccal surfaces of all teeth using one sterile Kerr applicator per arch. The samples were placed in 5 mL tubes containing glass beads and 1 mL of pre-reduced saline. The tubes were stored at room temperature and transported the same day from the schools to the laboratory by study staff. Samples were processed the same day or within 24 hours of collection. In the laboratory, plaque was prepared in pre-reduced saline and 10-fold dilutions were prepared separately. For each sample, a modified Mitis Salivarius agar (Difco Laboratories Inc., Detroit, MI, USA) supplemented with 500 μg/mL kanamycin, 1% potassium tellurite solution, and 50 U/mL bacitracin (MSKB) was used to enumerate S. mutans/sobrinus. The MSKB medium is more specific than MSB for MS isolation [10,11] but does not distinguish between S. mutans and S. sobrinus although a previous study found that more than 75% of the isolates were S. mutans [11]. Thus, counts from MSKB plates included both S. mutans and S. sobrinus and is denoted as S. mutans/sobrinus in this report to clearly show which species of MS are being referenced. Rogosa SL agar was use to culture and enumerate the Lactobacillus spp. [12]. Freshly prepared plates were used. The plaque samples were vortexed to break up the plaque and then diluted. The 10-0 to 10-3 dilutions were plated on MSKB media and incubated in 5% CO2 at 36.5°C for five to seven days. The Rogosa SL agar was incubated anaerobically at 36.5°C for seven days prior to enumeration.Statistical proceduresDescriptive statistics were used to describe the cohort. Significance of pre- to post-intervention reductions in bacteria levels within groups were assessed using paired T-tests. The analysis of covariance (ANCOVA) was used to assess the association between different treatment groups for pre- and post-intervention bacterial levels. The latter was adjusted for compliance. The SPSS (v.11.5) and SAS (v.9.1) statistical software were used.ResultsBaselineA total of 154 children participated in the three-arm study: 15.6 g xylitol/day (X16, n = 53); 11.7 g xylitol/day (X12, n = 49); and 44.7 g maltitol/day, (M45, n = 52). The mean age (standard deviation) of the children was 8.4 (1.4) years, 55% were boys (85/154) and 90% (138/154) was Caucasian. The mean age and racial/ethnic distribution among the groups were similar (Table 1). At baseline, 42 children (27.3%) did not have measurable S. mutans/sobrinus levels (< 102 cfu/mL. X16 = 17/52, X12 = 12/49, and M45 = 13/53) and 54 children (35.1%) did not have measurable Lactobacillus spp. (< 102 cfu/mL, X16 = 17/52, X12 = 19/49, and M45 = 18/53). Twenty nine (18.8%) children had S. mutans/sobrinus levels ≥ 104 (X16 = 21.1%, X12 = 22.4%, and M45 = 13.2%) and among these, 7 (4.5%) children had levels ≥ 106 (X16 = 2, X12 = 2, M45 = 3). The mean log10 S. mutans/sobrinus or Lactobacillus spp. levels in plaque between the groups at baseline were not statistically different. Excluding children who did not have measurable S. mutans/sobrinus and Lactobacillus spp. increased the baseline mean S. mutans/sobrinus s and Lactobacillus spp. levels, respectively; however, there were still no significant differences in mean reduction between the groups.Table 1Descriptive statistics and gummy bear compliance by group.GroupMaltitol 44.7 g/dayXylitol 11.7 g/dayXylitol 15.6 g/dayTotalN534952154Mean Age (SD)8.63 (1.4)8.01 (1.3)8.60 (1.37)8.42 (1.38)Male (%)26 (49%)31 (63%)28 (54%)85 (55%)Caucasian (%)49 (92%)42 (86%)47 (90%)138 (90%)% Missed GB (SD)21% (24.7)25% (28.1)24% (26.2)23% (26.3)Six week follow-upAt the six week follow-up, 97% of the children completed plaque sampling collection (X16 = 49/52, X12 = 48/49, M45 = 52/53). Overall, children consumed 77% of the total number of gummy bears possible for the study period (X16 = 76.4%, X12 = 73.7%, M45 = 80.4%, Table 1). Nearly all children consumed some gummy bears at each distribution session. However, 15% of the children were frequently absent and consumed less than 50% of their gummy bear allotment while several consumed less than 10% allotted. The proportions of these children were evenly distributed among the groups.The hypothesis tested was whether consumption of xylitol gummy bear snacks at the study dosages over a six-week period, including the one week ramp-up, would reduce S. mutans/sobrinus and Lactobacillus spp. levels in plaque. A reduction in bacteria levels in response to maltitol exposure was not expected. Results showed significant reductions in S. mutans/sobrinus levels pre- to post-intervention for the xylitol groups as well as for the maltitol group (group, log10 mean reduction [SD], p-value: X16, 1.13 [1.65], p < 0.0001; X12, 0.89 [1.11], p < 0.0001; and M45, 0.91 [1.46], p < 0.0001, Table 2 & Figure 2). Analysis of the covariance (ANCOVA) showed the reductions in S. mutans/sobrinus were not statistically different between the three groups or when xylitol groups (X16 + X12) were combined and compared to the maltitol group (Table 3). Thirty-eight children changed from detectable S. mutans/sobrinus to non-detectable levels (X16 = 13/52, X12 = 10/49, and M45 = 15/53). Two children, one from each from group X16 and X12, changed from non-detectable to detectable levels.Table 2Mean log10 S. mutans/sobrinus and Lactobacillus spp. levels and standard deviation at baseline, six weeks and the differences between six weeks and baseline for schoolchildren exposed to gummy bear snacks containing either xylitol (11.7 g/d or 15.6 g/d) or maltitol.GroupNVariableMeanStd DevPaired T-TestM45 (maltitol 44.7 g/d)53 S. mutans/sobrinusBaseline2.091.73Six weeks1.171.55Six weeks – baseline-0.911.46p < 0.0001 Lactobacillus spp.Baseline2.111.82Six weeks1.631.92Six weeks – baseline-0.522.41p = 0.13X12 (xylitol 11.7 g/d)49 S. mutans/sobrinusBaseline2.361.81Six weeks1.421.64Six weeks – baseline-0.891.11p < 0.0001 Lactobacillus spp.Baseline1.791.86Six weeks1.891.87Six weeks – baseline0.112.26p = 0.75X16 (xylitol 15.6 g/d)52 S. mutans/sobrinusBaseline2.252.00Six weeks1.131.62Six weeks – baseline-1.131.65 Lactobacillus spp.Baseline2.291.65p = 0.75Six weeks2.051.91Six weeks – baseline-0.312.35P = 0.36Figure 2Mean S. mutans/sobrinus levels in plaque at baseline and six weeks of gummy bear exposure. Mean S. mutans/sobrinus levels in plaque at baseline and six weeks for school children exposed to in-school gummy bear snacks containing either xylitol (11.7 g/d or 15.6 g/d) or maltitol three times per day.Table 3Analysis of the Covariate (ANCOVA) comparing S. mutans/sobrinus and Lactobacillus spp. levels at six weeks of gummy bear snack exposure between groups*Comparison GroupF-valueP-valueS. mutans/sobrinusMaltitol vs. Xylitol groups combined0.160.69X16 Xylitol vs. X12 Xylitol0.640.43X16 Xylitol vs. M45 Maltitol0.000.99X12 Xylitol vs. M45 Maltitol0.550.46Lactobacillus spp.M45 Maltitol vs. Xylitol groups combined0.130.72X16 Xylitol vs. X12 Xylitol0.000.99X16 Xylitol vs. M45 Maltitol0.090.76X12 Xylitol vs. M45 Maltitol0.100.75*Analysis of the Covariate (ANCOVA) adjusted for treatment assignment, baseline S. mutans/sobrinus or Lactobacillus spp. level, interaction term between baseline and treatment, and percent gummy bears missed.Changes in Lactobacillus spp. levels pre- to post-intervention were mixed with mean reduction (SD) observed for groups X16 = 0.31 (2.35) log10, and M45 = 0.52 (2.41) log10 but an increase in levels seen with X12 = 0.11 (2.26) log10, (Table 2). However, these mean changes were not significant and were within or near the measurement error, 0.5 log10. Adjusting for percent of unconsumed gummy bears did not significantly change the statistical results for either S. mutans/sobrinus or Lactobacillus spp.When children with no measurable S. mutans/sobrinus levels at baseline (n = 42) were removed from analyses, the mean log10 reduction (SD) for each group increased, X16 = 1.74 (1.67); X12 = 1.21 (1.10); M45 = 1.22 (1.57), and remained significant, P < 0.0001, (Table 4 & Figure 3). Comparison of reductions between the three groups remained non-significant.Figure 3Mean S. mutans/sobrinus levels in plaque at baseline and six weeks excluding children with non-measurable S. mutans/sobrinus levels at baseline. Mean S. mutans/sobrinus levels in plaque at baseline and six weeks for school children exposed to in-school gummy bear snacks containing either xylitol (11.7 g/d or 15.6 g/d) or maltitol three times per day after excluding 42 children with non-measurable S. mutans/sobrinus levels at baseline.Table 4Mean Log10 S. mutans/sobrinus and Lactobacillus spp. levels and standard deviation at baseline, six weeks and the differences between six weeks and baseline after excluding 42 children with non-measurable S. mutans/sobrinus levels at baseline for children exposed to xylitol (11.7 g/d or 15.6 g/d) or maltitol (44.7 g/day) gummy bear snack.GroupNVariableMeanStd DevPaired T-TestM45 (maltitol 44.7 g/d)40 S. mutans/sobrinusBaseline2.771.44Six weeks1.561.62Six weeks – baseline-1.221.57p < 0.0001 Lactobacillus spp.Baseline2.091.86Six weeks1.832.03Six weeks – baseline-0.312.40p = 0.42X12 (xylitol 11.7 g/d)37 S. mutans/sobrinusBaseline3.121.39Six weeks1.871.66Six weeks – baseline-1.211.10p < 0.0001 Lactobacillus spp.Baseline1.961.86Six weeks1.971.90Six weeks – baseline0.012.39p = 0.99X16 (xylitol 15.6 g/d)35 S. mutans/sobrinusBaseline3.351.49Six weeks1.611.75Six weeks – baseline-1.741.67p < 0.0001 Lactobacillus spp.Baseline2.511.85Six weeks2.411.94Six weeks – baseline-0.142.25p = 0.73Snack program acceptabilityPost-study informal discussions were held with school principals, teachers, and community workers. All agreed that it was feasible to implement an in-classroom xylitol gummy bear snacks program for caries prevention, and that parents are accepting of the program and children are willing to consume gummy bear snacks daily. Teachers indicated that although challenging initially, it was possible to organize classroom activities to incorporate the snack time, that the community workers were of tremendous value in keeping the program on track and flowing, and that the children were generally well behaved during snack time and followed directions once they became accustomed to the routine. Community workers enjoyed the interaction with the teachers and children and they liked the idea that they were contributing to a program that could improve children's oral health.DiscussionThis study is part of a series to explore effective xylitol delivery vehicles that can be used in school programs in the U.S. Results from the xylitol dose study [3] showed groups consuming 6.9 g and 10.3 g xylitol per day delivered via chewing gum had significant reductions in S. mutans/sobrinus levels in plaque and saliva after five weeks and six months of exposure. The smallest dose, 3.4 g/day, showed a small but not statistically significant reduction. However, the study did not have groups consuming doses between 3.4 g and 6.9 g per day, thus it is possible that doses within this range would also be effective. The xylitol frequency study [4] where the xylitol dose was kept constant (10.3 g/day) and frequency varied (0, 2, 3, and 4), showed a linear response in S. mutans/sobrinus reduction with increasing frequency of use. However, a minimum frequency of three administrations per day was required for xylitol chewing gum at therapeutic dose to significantly reduce S. mutans/sobrinus levels in plaque and saliva after five weeks of exposure. Twice pay day use group showed a small reduction consistent with the linear line model but was not statistically different from the control. These studies attempted to establish the minimum effective dose and frequency for the use of xylitol. This is important toward establishing guidelines for effective dose and frequency of xylitol use.Another study in this series assessed the bioavailability of xylitol in saliva over 15 to 20 minutes during and after consumption of different xylitol-containing products including: pellet (2.6 g) and stick (3 g) chewing gums, syrup (2.7 g), and gummy bear (2.6 g) [13]. Participants swallowed normally and at 10 to 11 specified time points, spat into a receptacle until a minimum of 0.5 mL saliva was collected. Xylitol concentration in saliva was measured by high performance liquid chromatography (HPLC). The results showed similar time-curves as well as areas under the curve for the products tested. Thus, similar dose and frequency of xylitol consumption via these products would likely result in comparable S. mutans/sobrinus reduction.In the present study, we tested the effectiveness of gummy bear snacks containing higher levels of xylitol in reducing S. mutans/sobrinus and Lactobacillus spp. levels in children's plaque after six weeks of exposure. The study was designed and implemented prior to initiation of the bioavailability study when uncertainty remained as to whether different xylitol-containing products would have comparable oral bioavailability. The study design controlled for frequency and number of gummy bears consumed. A school-based randomized controlled clinical trial currently underway is using a lower xylitol dose (7.8 g/day) and a natural fiber (inulin) gummy bear as the control with dental caries as an endpoint.The results showed that six weeks of habitual xylitol gummy bear consumption reduced the levels of S. mutans/sobrinus in plaque compared to baseline levels (Figure 2). A nearly one log10 mean reduction of S. mutans/sobrinus levels in children is highly significant given that children had lower levels of S. mutans/sobrinus infection (mean baseline ~2 log10) than among adults in our previous studies (mean baseline ~5 log10). The 15.6 g/day group had a slightly greater reduction in S. mutans/sobrinus levels but the difference was not significant compared to the 11.7 g/day group. This is in agreement with our dose-response study, which suggested a plateau effect at higher xylitol doses. Controlling for the number of unconsumed gummy bears among the groups did not change the results of within or across group comparisons. This supports findings from previous studies that xylitol reduces MS [14,15]. For a review of these studies, see Maguire & Rugg-Gunn (2003) [16]. More importantly, the results indicate that a gummy bear snack may be an effective method for delivering xylitol. However, these results should be interpreted cautiously given that a significant reduction in S. mutans/sobrinus levels was also observed with the maltitol group. Maltitol is a member of the sugar alcohol family that, as a class, is widely considered to be non-cariogenic. However, aside from xylitol, studies involving sugar alcohols, most commonly sorbitol, suggest they have little effect in actively reducing MS levels.There have been only a few studies on the association between maltitol and MS or dental caries. In a specific pathogen-free (SPF) Sprague-Dawley rats study, Ooshima and colleagues reported that maltitol did not induce dental caries in SPF rats infected with MS including S. mutans MT8148R or S. sobrinus 6715 strain, and replacement of the dietary sucrose content with maltitol resulted in a trend towards caries reduction in SPF rats [17]. However, a study conducted in Estonia found xylitol-maltitol and xylitol-polydextrose candies (49% xylitol in each) were equally effective in reducing MS suggesting that maltitol gave no added benefit to xylitol [18]. In a more recent study comparing xylitol (7.9 g/day) and maltitol (7.1 g/day) chewing gum to control (no gum) consumption over a six month period, Haresaku and colleagues reported that xylitol significantly reduced MS levels in plaque while maltitol increased MS levels [19]. This is clearly incongruous with our findings. However, the authors acknowledged several weaknesses in the study including a non-randomized study design (participants had the option to select their gum flavor which dictated their group assignment), average age of the maltitol group was higher which had a negative association with plaque MS levels, and only 59% of the control group remained in the final analysis. In addition, our study used a very high maltitol dose (44.7 g/day) 4–6 times that of previous studies, thus the results cannot aptly be compared nor can a direct comparison between the 15.6 or 11.7 g/day xylitol dose and the 44.7 g/day maltitol dose be made. Finally, it has been suggested that high or habitual polyols consumption may disturb polysaccharide synthesis leading to plaque and thus MS that are more loosely bound to teeth surfaces, consequently reducing MS levels [20]. Nevertheless, conflicting evidence exists and further research on the association between maltitol, S. mutans/sobrinus, and dental caries is needed.The finding that S. mutans/sobrinus reductions were not significantly different between the two xylitol groups was not surprising as our previous dosage study in this series suggested a plateau effect at higher therapeutic doses and our oral bioavailability study showed similar time curves and areas under the curve for xylitol chewing gums, syrup, and gummy bears. The reduction observed with maltitol was also not significantly different from the xylitol groups individually or combined. Although a systematic error in processing, culturing, or enumerating of S. mutans/sobrinus in the latter part of the study might explain the lower levels observed in all three groups, it would be unlikely since the reduction observed with the xylitol groups was as expected. Furthermore, sample processing, transport, culturing, and enumeration were carried out by protocol as was done with the baseline samples and the research and laboratory staff had remained unchanged, were highly experienced, and had carried out similar protocols in the previous studies of this series. The mechanical act of frequent chewing of gummy bears may reduce plaque formation and S. mutans/sobrinus adhesion but then Lactobacillus spp. levels should be similarly affected yet that was not observed. Lactobacillus spp. levels remained unchanged. Furthermore, chewing gum studies with sorbitol or sorbitol/maltitol gums have shown no effects on MS plaque or saliva levels [3,21,22]. Nevertheless, the xylitol results would have been strengthened if a true non-actively anticariogenic substance such as sorbitol was used as a control as in previous xylitol studies.As noted earlier, 27% of the children (X16 = 32%, X12 = 24%, M45 = 25%) did not have measurable S. mutans/sobrinus levels at baseline and may have biased the results. Sub-analyses with these children removed showed greater mean log10 reductions in S. mutans/sobrinus levels for all groups (X16 = 1.74 vs. 1.13, X12 = 1.21 vs. 0.89, M45 = 1.22 vs. 0.91). There were no significant differences between the three groups in the levels of reduction.This study also assessed Lactobacillus spp. response to xylitol exposure as the literature contains conflicting information. Juric and colleagues reported a reduction in these bacteria after two months of xylitol chewing gum exposure [23]. The Turku sugar studies [24] and the Belize xylitol study [21] with much longer follow-up periods also reported reductions in salivary Lactobacillus spp. On the other hand, in a study comparing the effects of chewing xylitol (5 g/day), sorbitol, and fructose gum over a four week period on S. mutans and Lactobacilli in plaque, Loesche and colleagues reported only xylitol significantly reduced S. mutans levels and there was no effect on the Lactobacilli levels for any gum [14]. Similarly, a recent study among young adults (21 to 24 years of age) reported no change in salivary Lactobacillus spp. in response to xylitol chewing gum consumption three times per day (6 g total) over a three week period but reported a significant reduction in salivary MS level [25]. The current study found that neither of the xylitol groups nor the maltitol group had an effect on Lactobacillus spp. level. It is possible that xylitol selectively affects and reduces S. mutans/sobrinus levels without altering Lactobacillus spp. levels, the other bacteria implicated in development of caries. Alternatively, the six weeks follow-up period of this study might not have been of sufficient length to detect the effects on Lactobacillus spp. Nevertheless, xylitol studies with dental caries as an end-point have repeatedly shown xylitol's effectiveness in caries reduction [26].Finally, the post-study discussion with school principals, teachers, and community workers suggested that it is feasible to implement a school-based xylitol gummy bear snacks program. Teachers were willing and able to incorporate the three times per day snack program into their classroom curriculum with minimal disruption after a brief adjustment period. Parents were willing to allow their children to participate in an oral health program and to consume gummy bear snacks at school to reduce tooth decay. Children were willing to eat most of the gummy bears provided at each distribution. A significant degree of commitment, cooperation, motivation, and effort from all parties involved was necessary to successfully implement and carry out the program. The information and experience gained from the current study were valuable in strategizing and developing the protocols used to implement and carry out the xylitol gummy bear snacks randomized clinical trial being conducted among kindergarten children in an elementary school district in Cleveland, Ohio. A lower xylitol dose is being used.This study had several limitations. Plaque collection was not standardized by plaque weight but rather by sampling the arches of teeth. This may be viewed as qualitative and may affect the quantitative analysis. However, the effect is minimized by standardization of sampling technique, by staff training, and by the use of staff with previous experience in this sampling technique. Furthermore, our previous studies with xylitol chewing gums showed that S. mutans/sobrinus levels in plaque sampling at screening were comparable to those at baseline for subjects and throughout the study among controls. The use of the manufacturer's (Santa Cruz Nutritionals) high dose maltitol (44.7 g) formula rather than the sorbitol/polydextrose formula was necessary because the sorbitol formula would not congeal properly to form the gummy bear during production. The reduction observed with high dose maltitol may have weakened our xylitol results. The inherent limitations of the setting and its capacity to deliver a public health intervention are an additional weakness. In this study, the intervention was delivered in a school setting and was subject to school closures and early dismissals, children's absences and cooperation, and classroom activities. For example, we experienced fluctuating cooperation of the children (e.g., some wanted to negotiate the number of bears to be consumed on some days, some preferred a specific color). Despite these difficulties, the local community workers who delivered the gummy bears made valiant attempts at getting the gummy bears to the children. Overall, 73% of the children consumed at least 75% of the total possible gummy bears distributed.Based on our findings, it is feasible to develop and implement xylitol-based caries preventive programs in structured settings such as schools and daycare. These structured environments offer an opportunity to achieve xylitol frequency and dose compliance. However, implementing a new activity into an institutional setting would undoubtedly be a challenging task and require the acceptance and commitment of all parties within the setting (e.g., school administration, teachers, parents and students, etc.).ConclusionXylitol gummy bear snack consumption at therapeutic dose and frequency reduced S. mutans/sobrinus but not Lactobacillus spp. levels in plaque after six weeks of habitual exposure. Xylitol doses in the high therapeutic range such as those in this study, 11.7 g and 15.6 g/day, produced similar reductions confirming a plateau of effect previously reported in this series of studies. High dose maltitol (44.7 g/day) also reduced S. mutans/sobrinus levels in plaque after six weeks of exposure. It is feasible to implement a xylitol gummy bear snack program in the school setting or daycare. Further study is needed to confirm the results and help determine if implementing such a program should be seriously considered to reduce the burden of dental caries among U.S. children, particularly those in high-risk populations.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsKAL was the project director and PM was the principal investigator for the overall series of xylitol studies. CAR coordinated this study. MR contributed to the design of this study, supervised and carried out the study procedures and contributed in the editing of the manuscript. MCR was the director of the laboratory that processed the studies' samples, generated all the numbers for S. mutans/sobrinus and Lactobacillus spp., and contributed to the studies' laboratory design and procedures. LZ was the data manager and analyst. All authors read and approved the final manuscript.Pre-publication historyThe pre-publication history for this paper can be accessed here:Supplementary MaterialAdditional file 1Gummy bear study informational school letter to parents. Letters sent to parent prior to recruitment with information about the study.Click here for fileAdditional file 2Gummy bear study baseline health questionnaire. Questionnaire used to obtain descriptive and general health information.Click here for file\n\nREFERENCES:\nNo References"
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+{
+    "id": "PMC2527578",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2527578\nAUTHORS: Qingwu Yang, Sing-Hoi Sze\n\nABSTRACT:\nBackgroundSince experimental determination of protein folding pathways remains difficult, computational techniques are often used to simulate protein folding. Most current techniques to predict protein folding pathways are computationally intensive and are suitable only for small proteins.ResultsBy assuming that the native structure of a protein is known and representing each intermediate conformation as a collection of fully folded structures in which each of them contains a set of interacting secondary structure elements, we show that it is possible to significantly reduce the conformation space while still being able to predict the most energetically favorable folding pathway of large proteins with hundreds of residues at the mesoscopic level, including the pig muscle phosphoglycerate kinase with 416 residues. The model is detailed enough to distinguish between different folding pathways of structurally very similar proteins, including the streptococcal protein G and the peptostreptococcal protein L. The model is also able to recognize the differences between the folding pathways of protein G and its two structurally similar variants NuG1 and NuG2, which are even harder to distinguish. We show that this strategy can produce accurate predictions on many other proteins with experimentally determined intermediate folding states.ConclusionOur technique is efficient enough to predict folding pathways for both large and small proteins at the mesoscopic level. Such a strategy is often the only feasible choice for large proteins. A software program implementing this strategy (SSFold) is available at .\n\nBODY:\nBackgroundAs early studies revealed that an unfolded protein can fold spontaneously to a three-dimensional structure under suitable environmental conditions [1,2], traditional approaches to understanding protein folding have focused on the prediction of the native structure. As more studies showed the existence of intermediates and interaction among residues during the protein folding process [3,4], there is substantial interest to understand the time order of events during the formation of the tertiary structure. From the free energy point of view, each conformation of a protein is associated with a free energy and the protein folds from the high-energy denatured conformation to its folded structure along a funnel-like energy landscape [5,6].Although advances in experimental techniques allow the investigation of protein folding pathways at the microsecond timescale [7,8], experimental determination of protein folding pathways remains difficult. Most studies are only able to identify general characteristics of the folding pathway without much details and are limited to analyzing small proteins. Computational techniques are often used to simulate protein folding and the problem is transformed to energetic optimization problems, that is, computational search for global energy minimum over all possible conformations. The most accurate computational techniques utilize molecular dynamics to determine the order of events that lead to the tertiary structure through atomic-level simulations [9-12]. Due to the extremely large conformation space, these approaches suffer from well-known problems accompanying high dimensionality, including computational expensiveness and ease of trapping in local minima, and are applicable only to small proteins in a short time course.By omitting some details, proteins can be represented at the level of amino acids. Kolinski and Skolnick [13] performed Monte Carlo simulations of protein folding on a reduced lattice representation of the protein α-carbon backbone. Yue and Dill [14] limited the conformation space to a discrete subset of possibilities and used a branch-and-bound procedure to search for near-optimal conformations. Alm and Baker [15] and Muñoz and Eaton [16] further observed that the availability of the known native structure can dramatically reduce the search space. Alm and Baker [15] took into account only native interactions among residues and used a sequential binary collision model to predict protein folding mechanisms from the perspectives of free energy landscapes, while Muñoz and Eaton [16] used a slightly different approach of employing distinct free energy costs for different secondary structures. Amato and Song [17] represented a protein by the torsional angles of its residues and used the probabilistic roadmap technique with a biased sampling strategy around the native structure to predict folding pathways and secondary structure formation order. Liwo et al [18] and Kmiecik and Kolinski [19,20] showed that the use of reduced models of proteins is highly successful in characterizing folding pathways for small proteins at the mesoscopic level. Although these techniques are able to predict folding pathways very accurately for proteins with up to about 100 residues, the majority of proteins in the Protein Data Bank (PDB) [21] are much larger (Figure 1).Figure 1The distribution of the number of atoms, the number of amino acid residues, and the number of secondary structure elements among 32237 protein structures in the Protein Data Bank (PDB) [21]. Each bar (except the rightmost one in each chart) shows the number of proteins that have values falling between the indicated lower and upper limits. The rightmost bar in each chart shows the number of proteins that have values of at least the indicated lower limit.The problem with representing a protein at the amino acid level is that even with the assumption that each residue has only two states (ordered or disordered), a protein with n residues still has 2n possible conformations [15]. To overcome this problem, several recent approaches represent a protein at the level of secondary structure elements (SSEs), in which each element corresponds to one helix or one β-strand. By adopting the framework model in which secondary structures are thought to fold relatively independently of the tertiary structure [22], each SSE is treated as an indivisible unit that interacts with other SSEs as a whole. Since the number of SSEs in a protein is small (Figure 1), this model is much more tractable to simulate. Eyrich et al [23] assumed that the SSEs are fixed and used a branch-and-bound algorithm to search for near-optimal tertiary structures. Apaydin et al [24] assumed that each SSE of a protein is already in native conformation and moves as a unit, and used the probabilistic roadmap approach to predict folding pathways. Zaki et al [25] proposed an algorithm to predict unfolding pathways based on applying a minimum cut procedure to a weighted graph that represents a protein's contact map or interaction strength between SSEs. Although the underlying assumption that intermediate secondary structures are fully folded before the formation of tertiary structures is not satisfied for most proteins, these studies show that such a strategy is sufficient to study protein folding pathways at the mesoscopic level.In this paper, our goal is to further reduce the conformation space without sacrificing prediction accuracy. This is achieved by assuming that SSEs that do not yet interact with each other are independent and can be treated separately. A conformation is represented by a collection of fully folded structures in which each of them contains a set of interacting SSEs. By using a steepest descent strategy, we show that it is possible to predict the most energetically favorable folding pathway of large proteins with hundreds of residues at the mesoscopic level and this model is detailed enough to distinguish between different folding pathways of structurally very similar proteins. In difference from the technique in [24], we do not consider the spatially moving process before the SSEs form native contacts, and thus we are able to achieve much better computational efficiency.MethodsAssume that the native structure of a protein is known. The protein folding pathway prediction problem is to find an ordered sequence of intermediate conformations to fill the gap between the unfolded state and the native tertiary structure. At the secondary structure level, a protein can be viewed as an ordered sequence of secondary structure elements (SSEs) interspersed with irregular turns or loops, where each SSE is either a helix or a β-strand, and each β-sheet consists of a variable number of β-strands that are not necessarily consecutive on the primary sequence. We represent each protein by t0s1t1⋯sktk, where k is the number of SSEs, si denotes the ith SSE, tj denotes the jth turn, and these elements are in the same order as they appear on the primary sequence. Given the three-dimensional structure of a protein, the assignment of SSEs can be obtained directly from the Protein Data Bank (PDB) [21] or using programs such as DSSP [26].Following [24] and [25], we consider each SSE as an indivisible unit that folds independently of the others according to the contacts present in the native structure. This is based on the framework model that assumes that extensive intermediate secondary structures exist before they are assembled into the tertiary structure [22], and our goal is to predict the interaction order of SSEs during folding. Based on the observation in [15] and [16] that a model using only native interactions can explain most experimental results, we assume that the interactions between SSEs or turns are the same as the ones present in the native structure. Although these assumptions are often not satisfied as there are many proteins in which there are no clear secondary structures before the formation of tertiary structures or there are no clear preservations of secondary structures throughout folding, such a strategy is sufficient for studying folding pathways at the mesoscopic level and is often the only feasible choice for large proteins.We represent a conformation of a protein on the folding pathway by C = {S1, ..., Sk}, where each Si represents a structure consisting of a set of fully folded SSEs and there are no interactions between two different sets Sj and Sj' (see Figure 2 for an illustration). Since our focus is on the SSEs, turns are not included in the conformation but will be utilized when computing energies (see below). The protein folding problem is transformed to identifying a sequence of conformational changes that start from an initial state with fully folded SSEs but no interactions between SSEs through some intermediate conformations and ending in the native structure (Figure 2). Each conformational change corresponds to finding a new pair of interactions that merges two smaller structures of SSEs into a bigger one. Figure 2 illustrates the folding pathway prediction on the B1 domain of the streptococcal protein G (GB1). In the prediction, β3 and β4 interact first, then α1 is added, followed by β1 and β2.Figure 2Illustration of the folding pathway prediction for GB1. The starting conformation {{β1}, {β2}, {α1}, {β3}, {β4}} corresponds to the initial state. There are three intermediate conformations in the predicted folding pathway, including {{β1}, {β2}, {α1}, {β3, β4}}, {{β1}, {β2}, {α1, β3, β4}}, and {{β2}, {β1, α1, β3, β4}}. The ending conformation {{β1, β2, α1, β3, β4}} corresponds to the native state.Folding pathway predictions are obtained through the computation of free energies of intermediate conformations. For an intermediate conformation C = {S1, ..., Sk}, the free energy E(C) of C is defined as:E(C)=∑i=1kE(Si),where each Si is viewed as an isolated entity and each E(Si) is obtained separately by extracting the three-dimensional coordinates of its residues from the Protein Data Bank (PDB) [21] and using the Rosetta software [27] to compute its free energy. The original Rosetta energy function is used, which is obtained by representing each side chain by a centroid that is located at the center of mass, and computing a weighted sum of the binned probability descriptions of multiple effects, including the solvation and electrostatic effects based on observed distributions in known protein structures, the secondary structure packing effects that include strand pairing, strand arrangement into sheets and helix-strand packing, and the effects of steric repulsion and Van der Waals interactions (more details are available in [28] and in Table I of [27]). To take the backbone into consideration, a turn tj is included in the computation of E(Si) if both of its adjacent SSEs sj (if it exists) and sj+1 (if it exists) are included in Si.Since the interactions that favor folding usually decrease the free energy while the interactions that destabilize the native structure increase the free energy, our goal is to find the most energetically favorable folding pathway by identifying the conformational change that decreases the free energy the most in each step so that the protein can get to lower energy states as quickly as possible. Figure 3 illustrates our SSFold algorithm that uses a steepest descent strategy to choose a new pair of interactions that leads to a conformation with the lowest free energy in each iteration. This procedure is very efficient since only k - 1 iterations are needed. Within each iteration, O(k2) comparisons are needed to find the best pair of interactions that results in the lowest free energy. This leads to an overall time complexity of O(k3t), where k is the number of SSEs in a protein and t is the time to compute the free energy of a potentially partial protein that contains only some of the SSEs and turns.Figure 3Algorithm SSFold to predict the most energetically favorable interaction order of SSEs that corresponds to a folding pathway. Each iteration corresponds to a conformational change that results from a new pair of interactions. Within a folded structure, a turn is included in the energy computations only when adjacent SSEs are included in the structure.ResultsWe test our strategy on proteins from the Protein Data Bank (PDB) [21] that have known intermediate folding states from experimental data. We illustrate that our model is detailed enough to distinguish between subtle differences in the folding pathways of the streptococcal protein G, the peptostreptococcal protein L, and variants NuG1 and NuG2 of protein G, which are all structurally very similar proteins. We demonstrate that our approach is applicable to large proteins with hundreds of residues by testing it on the 416 residue pig muscle phosphoglycerate kinase (PGK). We further test it on proteins studied in [29] and [25] to validate that our model has very good accuracy.Proteins GB1, LB1, NuG1 and NuG2The 56 residue B1 immunoglobulin binding domain of streptococcal protein G (GB1, PDB: 1GB1) and the 62 residue B1 immunoglobulin binding domain of peptostreptococcal protein L (LB1, PDB: 2PTL) have been used extensively as model systems for studying protein folding mechanisms [30-37]. Both GB1 (see Figure 2) and LB1 consist of one β-sheet with four strands and one α-helix. Strands 1 and 2 form an N-terminal β-hairpin, while strands 3 and 4 form a C-terminal β-hairpin. Although GB1 and LB1 have very similar tertiary structures, they have different folding pathways. As suggested by [29], a detailed model is needed to distinguish between them.Figure 4 shows our folding pathway predictions for GB1 and LB1 (see also Figure 2 for GB1).Figure 4Folding pathway predictions for GB1, LB1, NuG1 and NuG2. Each internal node represents a new pair of interactions and nodes that are higher in the tree indicate earlier interactions. Also compare to Figure 2 for GB1.Experimental results showed that the C-terminal β-hairpin in GB1 is formed in the transition state of the folding pathway and serves as the starting point on which the rest of the protein can fold [35]. Similar results were obtained using the diffusion-collision model [38]. Our prediction is consistent with these results. In contrast, experimental results showed that only the N-terminal β-hairpin in LB1 is mainly formed in the transition state and non-random structures can be detected in the region [34,39]. Our algorithm also predicts that the N-terminal β-hairpin forms earlier than the C-terminal β-hairpin in LB1.Two protein G variants, NuG1 (PDB: 1MHX) and NuG2 (PDB: 1MI0), were designed to have a different folding mechanism from protein G by replacing some residues of protein G [36]. In NuG1 and NuG2, the stability of the N-terminal β-hairpin is enhanced while the stability of the C-terminal β-hairpin is reduced, with the N-terminal β-hairpin forming contacts earlier than the C-terminal β-hairpin in both cases [36].Thomas et al [40] showed that it is more difficult to distinguish between the folding pathways of protein G and its variants NuG1 and NuG2 than to distinguish between the folding pathways of protein G and protein L. In our predictions in Figure 4, NuG1 and NuG2 have the same folding pathway, with the N-terminal β-hairpin folded first. This is consistent with the experimental results in [41] and the predictions in [40].Figure 5 shows the free energy profiles of GB1, LB1, NuG1 and NuG2 in our predictions. Our predicted folding pathway of GB1 is a non-frustrated curve, similar to the average macroscopic folding pathway given by [37]. When compared to GB1, NuG1 and NuG2 have similar profiles and higher initial free energy, but their native structures have lower free energy and are more stable, which is consistent with the analysis in [41].Figure 5Free energy profiles of GB1, LB1, NuG1 and NuG2 in our predictions. A native contact is defined to be a pair of amino acids that have their α-carbon atoms within 7 Å of each other. Each starting point corresponds to the initial state in which each SSE has already completed its native fold independently and there are no interactions between SSEs.Pig muscle PGK: a large proteinPhosphoglycerate kinase (PGK) from various organisms has been used as a model system for studying domain-domain interactions of multiple-domain proteins [42-44]. The pig muscle PGK (PDB: 1KF0) [43] is a large two-domain protein with 416 residues, with the N-terminal domain consisting of residues 1 to 155 and the C-terminal domain consisting of residues 156 to 416. There are 21 α-helices and 17 β-strands, which belong to four different β-sheets A, B, C and D, arranged as follows on the primary sequence: α1 βA4 α2 α3 βA3 α4 βA1 α5 βA2 α6 βB1 βB2 α7 βA5 α8 α9 βA6 α10 βC3 α11 α12 βC2 α13 α14 α15 βC1 βD2 βD1 βD3 α16 βC4 α17 α18 βC5 α19 βC6 α20 α21.Figure 6 shows our folding pathway prediction for the pig muscle PGK, in which β-sheet D is formed first, followed by the formation of β-sheet C interspersed with α-helices in the C-terminal domain. After most SSEs of the C-terminal domain are formed, the SSEs of the N-terminal domain begin to form, with β-sheet A formed before β-sheet B interspersed with α-helices in the N-terminal domain.Figure 6Folding pathway prediction for the pig muscle PGK.Szilágyi and Vas [45] suggested a sequential domain refolding mechanism for the pig muscle PGK, in which folding of the C-terminal domain is independent of the N-terminal domain and takes place first, and folding of the N-terminal domain starts after most of the C-terminal domain folds. The authors also suggested that an intermediate consists of a folded C-terminal domain and a still unfolded N-terminal domain. Our prediction is consistent with these experimental results.Other proteinsFigure 7 shows folding pathway predictions for various small proteins that have known intermediate folding states from biological experiments. The proteins 1BDD and 2CRT were studied in [29], while the proteins 1BIN, 1MBC, 2CI2 and 6PTI were studied in [25].Figure 7Folding pathway predictions for Staphylococcus aureus protein A domain B (PDB: 1BDD), leghemoglobin A (PDB: 1BIN), myoglobin (PDB: 1MBC), chymotrypsin inhibitor 2 structure 1 (PDB: 1COA), chymotrypsin inhibitor 2 structure 2 (PDB: 2CI2), cardiotoxin III (PDB: 2CRT), and bovine pancreatic trypsin inhibitor BPTI (PDB: 6PTI).The B domain of Staphylococcus aureus protein A (PDB: 1BDD) consists of three α-helices. In our prediction, α2 and α3 interact first, then α1 is added. This is consistent with the result of the out-exchange experiment in [46] and experimental results under high temperature [47].Although two members of the globin protein family, leghemoglobin A (PDB: 1BIN) and myoglobin (PDB: 1MBC), have very low sequence similarity, they both consist of eight α-helices and have very similar tertiary structures. Nishimura et al [48] compared their folding pathways experimentally. For leghemoglobin A, αG, αH, and part of αE form stable structures first, while αA and αB form in the later stages of the folding pathway. For myoglobin, αA, αG and αH form stable contacts first. The main difference between the two folding pathways is that αA and αB form earlier in the folding pathway of myoglobin than in the folding pathway of leghemoglobin A [48]. Our predictions are able to distinguish between these subtle differences. For leghemoglobin A, αG and αH are predicted to interact first, then αE is added, with αB and αA added later. For myoglobin, αG and αH are also predicted to interact first, then αA is added, followed by αE and αB.There are two crystal structures for chymotrypsin inhibitor 2 (PDB: 1COA and 2CI2). While 2CI2 consists of 83 residues, 1COA is a fragment of 2CI2 from residues 20 to 83. They both consist of one α-helix and four β-strands, which are arranged as β1α1β2β3β4 in 1COA and β1α1β4β3β2 in 2CI2. In our predictions, 1COA and 2CI2 have the same folding pathway, with the middle two β-strands interacting first, then the α-helix is added, followed by the C-terminal β-strand, and the N-terminal β-strand is added last. For 1COA, simulation by [49] demonstrated that β2 and β3 form contacts first, then α1 is added to form a folding nucleus. The coalescence of β1 is the rate-limiting step and is completed at the end of the folding process. This is consistent with the result of the out-exchange experiment in [46] that showed that β2, β3 and α1 form contacts first. Our prediction is consistent with these results.The all β-sheet protein cardiotoxin III (PDB: 2CRT) consists of five strands. While β1 and β2 form a double-stranded domain, β3, β4 and β5 form a triple-stranded domain. By the amide proton pulse exchange experiment, Sivaraman et al [50] showed that the triple-stranded domain forms earlier than the double-stranded domain during the refolding process. The carbonyl groups in β3 and the amide groups in β5 form hydrogen bonding partners, which are important for the formation of a hydrophobic cluster [50]. Our prediction is consistent with these results, with β3 and β5 interacting first, then β4 is added to form the triple-stranded domain, followed by β2 and β1 in the double-stranded domain.Bovine pancreatic trypsin inhibitor BPTI (PDB: 6PTI) is a globular protein with two α-helices and three β-strands, which are arranged as α1β2β1β3α2. Three disulfide bonds between residues 5 and 55, 14 and 38, and 30 and 51 play an important role in stabilizing the native structure [51], and their formation order was studied in [52]. In our prediction, β1 and β2 interact first, then α2 is added. This brings residues 30 and 51 close together and helps to form the disulfide bond between them. Then α1 is added and this helps to form the disulfide bond between residues 5 and 55, and 14 and 38. Our prediction that β1 and β2 interact earlier than the two α-helices is consistent with the result in [53].DiscussionWhile our strategy corresponds most closely to the diffusion-collision model that allows folding to proceed independently in different parts of a protein [54], it is possible to use a modified strategy for other models. For example, to simulate the nucleation-propagation model [55] or the nucleation-condensation model [56], in which the existence of a nucleus facilitates further folding, one can iteratively add a SSE that results in the lowest free energy to the nucleus. Since energy computations can still be slow and can take hours, which account for significant amount of computation time in our algorithm, it is also possible to use lower resolution methods to compute energy.While our strategy finds the most energetically favorable protein folding pathway, there are evidences that multiple folding pathways exist [5,57]. The ability to analyze multiple folding pathways will also allow the study of protein misfolding [58]. Our approach can be generalized to study the entire free energy landscape [5] as follows: construct a graph in which each vertex represents a biologically plausible conformation and each edge represents a feasible conformation change, which is similar to the roadmap graph in [24] and [17] and the protein folding network in [59] except that we consider each SSE as an indivisible unit. Various graph-theoretic algorithms can then be used to generate predictions of alternative folding pathways.ConclusionWe have shown that our procedure has sufficient accuracy to distinguish between subtle differences and our strategy can be applied to large proteins due to its speed. An important future direction is to consider cooperative folding of secondary structures without too much sacrifice in speed, that is, when folding in one secondary structure affects folding in others.Authors' contributionsQY performed the research and implemented the algorithm. S–HS supervised the research. All authors read and approved the final manuscript.\n\nREFERENCES:\nNo References"
+}

batch_11/PMC2527607.json

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+{
+    "id": "PMC2527607",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2527607\nAUTHORS: Nikolaos Barbetakis, Georgios Samanidis, Dimitrios Paliouras, Ioannis Boukovinas, Christos Asteriou, Eleni Stergiou, Kostas Laschos, Christodoulos Tsilikas\n\nABSTRACT:\nBackgroundAmong human neoplasms thymomas are associated with highest frequency with paraneoplastic autoimmune diseases.Case presentationA case of a 42-year-old woman with paraneoplastic pemphigus as the first manifestation of thymoma is reported. Transsternal complete thymoma resection achieved pemphigus regression. The clinical correlations between pemphigus and thymoma are presented.ConclusionOur case report provides further evidence for the important role of autoantibodies in the pathogenesis of paraneoplastic skin diseases in thymoma patients. It also documents the improvement of the associated pemphigus after radical treatment of the thymoma.\n\nBODY:\nIntroductionParaneoplastic pemphigus is rarely associated with thymic neoplasms either alone or concomitantly with other autoimmune disorders. A case of a 42-year-old woman with paraneoplastic pemphigus as the first manifestation of thymoma is reported. Transsternal complete thymoma resection achieved pemphigus regression. The pathogenetic and clinical relationships between pemphigus and thymoma encountered in this case are presented with the purpose of strengthening the hypothesis that pemphigus in the presence of a thymoma could be a thymoma-associated autoimmune disease.Case presentationA 42-year-old otherwise healthy woman was admitted to our hospital with numerous crusted, denuted and flaccid vesiculobullous lesions over her chest, abdomen, legs, toes and forehead (Figures 1, 2). Vesicles were also observed on the buccal mucosa and palate. The clinical diagnosis of pemphigus vulgaris was confirmed by biopsy. Histological findings included interface dermatitis, apoptotic keratinocytes and focal areas of suprabasal acantholysis. Therapeutic management consisted of successive cycles of 50 mg/day of prednisone. Medical treatment provided only transient relief of symptoms and temporary reduction in clinical manifestations and on interruption of systemic steroid the lesions reccured.Figure 1Numerous crusted, denuted and flaccid vesiculobullous lesions over the chest and abdomen consistent with pemphigus.Figure 2Close view of a pemphigus lesion.Three months later while facing a persistent pemphigus, a chest x-ray was done and a retrosternal tumor was noted (Figure 3). CT scans and MRI of the chest revealed a homogeneous anterior mediastinal mass consistent with a thymic tumor but with no lymph node involvement (Figure 4). In order to perform preoperative staging of the tumor, the patient underwent CT scans of brain, abdomen and a bone scan. All were normal. An autoimmune laboratory profile proved that desmoglein-1 (anti-DSG-1) antibodies were normal while the desmoglein-3 (anti-DSG-3) titer reached twice the normal.Figure 3A profile chest x-ray showing a retrosternal tumor.Figure 4MRI of the chest revealed an anterior mediastinal mass consistent with a thymic tumor.At operation through a median sternotomy, a solid intrathymic capsulated mass was found and a thymectomy was performed. Definitive pathologic examination found a tumor measuring 6 × 5 × 5 cm occupying the inferior part of the thymus gland consisting of spindle cells populated by varying numbers of lymphocytes with rounder epithelial cells (Figures 5, 6). No capsular involvement was noted (Type A non invasive thymoma according to World Health Organization classification).Figure 5Thymic tumor consisting of spindle cells.Figure 6Varying numbers of lymphocytes with rounder epithelial cells populating spindle cells consisting with a non invasive thymoma.The postoperative period was uneventful. The patient was discharged home after 10 days Postoperatively anti-DSG-1 and anti-DSG-3 showed a transient increase. At the outpatient visit four weeks later both desmoglein antibodies showed a significant decrease associated with almost complete resolution of the cutaneous lesions. Twelve months later she is still disease free and with no need of steroids.DiscussionThe relation between thymus disease and autoimmune disorders is well known and established [1]. In 1987 a retrospective study among 172 patients with thymoma found fungal mucocutaneous disease as the most common thymoma-associated cutaneous disorder [2]. Two patients in that study were noted with pemphigus and lichen planus respectively. Since then paraneoplastic pemphigus has been recognized as a well defined autoimmune syndrome characterized by autoantibody formation directed against epithelial antigens [3]. In our case pemphigus was diagnosed first by biopsy (Figure 7) and this has led us to further diagnostic investigation.Figure 7Microscopic appearance of pemphigus with epithelial cells falling away from each other and rounding out in the fluid of the blister.Serial analysis of various case reports correlate pemphigus with thymoma and/or myasthenia gravis although in our case there were no symptoms or signs of myasthenia gravis [4,5]. The clinical course and titrations of antiepithelial, antimuscle, and antithymus antibodies suggested a reverse relationship between the severity of myasthenia gravis and titers of antimuscle and antithymus antibodies, and a parallel relationship between pemphigus vulgaris and antiepithelial antibody.The association of paraneoplastic pemphigus with malignancy is strong. Only a handful of patients have had no associated diagnosis. Some patients have had benign neoplasms, including as in our case thymoma or Castleman's disease. Only a single patient without a tumor has met the diagnostic criteria, yet this patient had a rapid demise and may have died with an undiagnosed malignancy [6]. Patients have developed paraneoplastic pemphigus while in remission of their malignancy, leading some authors to prefer the term neoplasia-induced pemphigus.Treatment of the underlying malignancy does not necessarily halt progression of the paraneoplastic pemphigus, although some have observed that clinical manifestations improve as autoantibody titers decrease following resection of the tumor. This was also proved in our case, where anti-DSG-3 showed a significant decrease associated with almost complete resolution of the cutaneous lesions. Circulating and tissue-bound antibodies in patients with this disease are directed against a group of molecules with sequence homology and belonging to the plakin family. These molecules are found in the intracellular attachment plaques of desmosomes and hemidesmosomes, and they play a key role in intermediate filament attachment. However, the number of reported target antigens has increased over time and varies between patients. This variability likely accounts for the clinical heterogeneity of this disease. By immunoprecipitation, target antigens (in decreasing order of incidence) include desmoglein 3, desmoglein 1, envoplakin (210 kd), periplakin (190 kd), desmoplakin I (250 kd), desmoplakin II (210 kd) and bullous pemphigoid antigen I (230 kd). Plectin (> 400 kd) and an unidentified 170-kd protein have also been found [7].In our case the levels of pemphigus-associated antibodies had increased postoperatively after thymectomy. In parallel, decreased titer of those antibodies preceded the resolution of cutaneous lesions. A similar beneficial effect of tumor ablation has recently also been reported in the case of Castleman's tumor, a rare lymphoproliferative disease that is sometimes associated with paraneoplastic pemphigus [8].Conclusionour case report provides further evidence for the important role of autoantibodies in the pathogenesis of paraneoplastic skin diseases in thymoma patients. It also documents the improvement of the associated pemphigus after radical treatment of the thymoma. Further studies are necessary to analyze possible pathogenetic mechanisms.Conflict of interestsThe authors declare that they have no competing interests.Authors' contributionsNB, GS, DP, IB, CA, ES and KL took part in the care of the patient and contributed equally in carrying out the medical literature search and preparation of the manuscript. CT participated in the care of the patient and had the supervision of this report. All authors approved the final manuscript.\n\nREFERENCES:\nNo References"
+}

batch_11/PMC2527655.json

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+{
+    "id": "PMC2527655",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2527655\nAUTHORS: Howard Chia-Hao Chang, Douglas N. Dimlich, Takakazu Yokokura, Ashim Mukherjee, Mark W. Kankel, Anindya Sen, Vasanthi Sridhar, Tudor A. Fulga, Anne C. Hart, David Van Vactor, Spyros Artavanis-Tsakonas\n\nABSTRACT:\nSpinal Muscular Atrophy (SMA), a recessive hereditary neurodegenerative disease in humans, has been linked to mutations in the survival motor neuron (SMN) gene. SMA patients display early onset lethality coupled with motor neuron loss and skeletal muscle atrophy. We used Drosophila, which encodes a single SMN ortholog, survival motor neuron (Smn), to model SMA, since reduction of Smn function leads to defects that mimic the SMA pathology in humans. Here we show that a normal neuromuscular junction (NMJ) structure depends on SMN expression and that SMN concentrates in the post-synaptic NMJ regions. We conducted a screen for genetic modifiers of an Smn phenotype using the Exelixis collection of transposon-induced mutations, which affects approximately 50% of the Drosophila genome. This screen resulted in the recovery of 27 modifiers, thereby expanding the genetic circuitry of Smn to include several genes not previously known to be associated with this locus. Among the identified modifiers was wishful thinking (wit), a type II BMP receptor, which was shown to alter the Smn NMJ phenotype. Further characterization of two additional members of the BMP signaling pathway, Mothers against dpp (Mad) and Daughters against dpp (Dad), also modify the Smn NMJ phenotype. The NMJ defects caused by loss of Smn function can be ameliorated by increasing BMP signals, suggesting that increased BMP activity in SMA patients may help to alleviate symptoms of the disease. These results confirm that our genetic approach is likely to identify bona fide modulators of SMN activity, especially regarding its role at the neuromuscular junction, and as a consequence, may identify putative SMA therapeutic targets.\n\nBODY:\nIntroductionSpinal Muscular Atrophy (SMA) is the second most common autosomal recessive genetic disease in humans and is the leading cause of genetically linked infant mortality, with an incidence rate of approximately 1 in 6000 births [1], [2], [3]. Clinical manifestation of SMA shows degeneration of spinal cord motor neurons and muscle atrophy [4]. SMA has also been linked to two nearly identical genes located on chromosome 5, survival motor neuron 1 (SMN1) and survival motor neuron 2 (SMN2) [5]. SMN2 differs from SMN1 in that only 10% of SMN2 transcripts produce functional Smn protein (SMN) due to a mutation that results in its aberrant splicing [6], [7], [8].Elegant biochemical studies established the importance of the SMN protein in a ubiquitous, multimeric complex involved in the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs) [9], [10], [11], [12], [13], [14]. Despite its seemingly fundamental and indispensable role in cellular metabolism, reduction of SMN leads to a specific neurodegenerative profile associated with this disease [1], [15], [16], [17], [18]. Though several recent studies indicate that SMN influences motor neuron axonal morphology [19], [20], it remains unclear whether SMN has a specific neuromuscular junction (NMJ) function, and whether the functional requirement for SMN activity is increased at the NMJ than elsewhere in the organism.SMA results from loss of SMN1 function [6], [21], however, the clinical severity of the disease correlates with SMN2 copy number, which varies between individuals [22]. As the small amount of functional SMN2 protein produced by each copy is capable of partially compensating for the loss of the SMN1 gene function, higher copy numbers of SMN2 result in generally milder forms of SMA. Given that the severity of SMA depends on the levels of functional SMN, genetic modifiers capable of altering SMN cellular activity may define useful therapeutic targets. This reasoning prompted us to explore the genetic circuitry capable of affecting SMN activity in Drosophila, an experimental model amenable to sophisticated genetic manipulations, to investigate the role of SMN in this system.The Drosophila genome harbors a single copy of the Smn gene, which encodes a highly conserved homologue of SMN. The Smn loss of function allele, Smn\n73Ao, results in recessive larval lethality and, importantly, neuromuscular junction abnormalities [15], [18], [23]. In this study, we characterized additional Smn alleles and demonstrate that they also display NMJ defects. To analyze tissue-specific requirements of SMN, we used RNA interference (RNAi) to create a series of loss of function Smn alleles, whose phenotypes mimic the dosage dependent nature of SMA pathology. By using muscle (mesoderm) and neuronal drivers to direct expression of the Smn RNAi constructs, we determined that SMN function is required in both tissues, though there appears to be a higher sensitivity to the loss of SMN function in the muscle.To identify enhancers and suppressors of SMN activity and the genetic circuitry of Smn, we carried out a genetic screen for modifiers of the Smn\n73Ao allele using the Exelixis collection of insertional mutations, which affects approximately 50% of the Drosophila genome [24], [25], [26]. Of the 17 enhancers and 10 suppressors uncovered by the screen, a significant subset was shown to be capable of affecting Smn-related NMJ phenotypes, validating our approach. Amongst these Smn modifiers was wishful thinking (wit), which encodes a type II BMP receptor [27], [28]. Further experiments defined genetic interactions between Smn and other members of the BMP signaling pathway. We also demonstrated that modulation of BMP signaling rescues Smn-related NMJ phenotypes, further validating this genetic approach as a means to identify novel targets of SMN function. Moreover, it seems likely that some of the novel targets may provide potential therapeutic value.ResultsSMN concentrates in the post-synaptic regions at the NMJThe dichotomy between the ubiquitous housekeeping function of Smn and the very specific neuromuscular SMA phenotype raises the question whether Smn functions differently at the neuromuscular junction (NMJ) than in other tissue types. Specifically, whether SMN has a differential expression pattern in neurons and muscle and whether SMN concentrates to any particular cellular compartments at the NMJ remain open questions.To determine in which tissue(s) SMN is expressed in Drosophila we raised antibodies against full-length Drosophila SMN (See Materials and Methods) and monitored its expression pattern particularly at the NMJ. In Western blots performed on lysates derived from S2 cells, 3rd instar larvae and wild-type adult heads the antibody recognizes a single ∼28 kD band [18], corresponding to the predicted molecular weight of Drosophila SMN (Figure S1 and data not shown). Moreover, when a FLAG-tagged Smn transgenic construct (UAS-FLAG-Smn) was expressed under the control of the vestigialGAL4 driver, SMN and FLAG staining overlapped at the dorsal-ventral (DV) boundary of 3rd instar larval wing discs. In addition, vestigialGAL4-directed expression of an inducible RNAi allele of Smn (see below) abolished the SMN staining pattern along the DV boundary of the larval wing disc (Figure S1). Together, these results indicate the specificity of the antibody we raised against SMN.Using this antibody we probed SMN expression at the NMJ and found antigens to be clearly concentrated at the post-synaptic regions in the muscle, co-localizing with the post-synaptic marker Discs Large (DLG) (Figure 1A–D) [29]. Under these conditions, we did not detect antigens in the pre-synaptic region of the motor neuron terminal (as defined by horseradish peroxidase (HRP) staining) at the NMJ (Figure 1A–D). SMN staining was also observed within muscle fibers and at discrete foci in muscle nuclei (Figure 1C and E), which presumably reflect SMN localization in Cajal bodies (gems) as demonstrated for mammalian cells [9], and in Drosophila ovarian nurse cells and oocytes [30]. This post-synaptic NMJ expression pattern of SMN is abolished by muscle-specific Smn RNAi knockdown, again demonstrating the specificity of the anti-SMN antibodies (Figure S2). Consistent with its general role in snRNP assembly, SMN was detected in all tissues examined, including muscle (Figure 1A–D) and neurons (Figure 1F). However, at the Drosophila NMJ, SMN is concentrated at the post-synaptic regions in the muscle.10.1371/journal.pone.0003209.g001Figure 1SMN localizes to the post-synaptic region of the Drosophila NMJ.(A–D) SMN expression at the NMJ between muscle fibers 6 and 7. (A) Pre-synaptic anti-HRP staining (red), (B) post-synaptic anti-DLG staining (blue), (C) anti-SMN staining (green) and (D) a merge of (A–C). SMN expression co-localizes with DLG at the post-synaptic region of the NMJ. (E) SMN staining is also observed in muscle fibers and discrete foci in nuclei (arrow). (F) Though no pre-synaptic SMN staining is observed, robust levels of SMN expression are seen in the larval brain. Scale bars in (D), (E), (F) represent 10 µm, 20 µm, and 50 µm.Mutations in Smn compromise viabilityPrevious studies determined that loss of Smn function results in larval lethality [15], [18]. We examined two additional Smn alleles found within the Exelixis collection, Smn\nf01109 and Smn\nf05960\n[25], [26]. Sequence analysis of both strains indicates each allele harbors a transposon insertion within the Smn coding region (at amino acids I93 for Smn\nf01109 and K136 for Smn\nf05960, see Figure 2A) that is predicted to introduce a premature stop codon. (Figure 2A). Unlike the Smn\n73Ao allele [15], [18], which is 100% lethal in homo- and hemizygous (Smn\n73Ao/Df(Smn)) backgrounds (Figure 2B), the Smn\nf05960 allele produces a small percentage of escapers (3.3%) when mutant larvae are isolated and cultured at low density. On the other hand, Smn\nf01109 allele is semi-viable (67.7%) (Figure 2B), indicating that the Smn\nf01109 and Smn\nf05960 alleles are not null mutations as previously suggested [18]. By examining the viability of various Smn allelic combinations (Figure 2B), we determined that Smn\nf01109 is weakly hypomorphic as it retains some degree of viability in all cases tested, while Smn\nf05960 appears to act as a strong loss-of-function allele since it fails to complement both Smn\n73Ao and a small deficiency that uncovers Smn, Df(3L)Smn\nX7 (Figure 2B). Ubiquitous (tubulinGAL4, actinGAL4) expression of UAS-FLAG-Smn rescued Smn\nf05960 lethality, demonstrating the lethality was associated with a loss of Smn activity. This is consistent with earlier studies showing ectopic SMN expressed under the control of a ubiquitous driver (tubulinGAL4) rescued Smn\n73Ao lethality [15].10.1371/journal.pone.0003209.g002Figure 2\nSmn mutations cause lethality.(A) Schematic representation of the SMN protein and location of mutations corresponding to the Smn alleles used in this study. The conserved Tudor domain and YG box are indicated. Insertion sites of the transposon induced Smn\nf05960 and Smn\nf01109 alleles are denoted by triangles. Regions of the Smn transcript targeted by RNA interference (RNAi) are illustrated as lines under the SMN protein schematic. (B) Loss of Smn function elicits lethality. For individuals of given phenotypes, the percentages of surviving individuals are shown and are normalized to wild-type. Smn\n73Ao and Smn\nf05960 homozygotes die during late 2nd/early 3rd larval and pupal stages, though some Smn\nf05960 escapers are detected. In contrast, 67% of the Smn\nf01109 homozygotes survive to adulthood. Smn\nf01109/Smn\n73Ao and Smn\nf05960/Smn\n73Ao trans-heterozygous combinations are also viable. In addition, a small deficiency uncovering the entire Smn transcript was generated (Df(3L)Smn\nX7). We crossed all three Smn alleles to Df(3L)Smn\nX7 and found that both Smn\n73Ao/Df(3L)Smn\nX7 and Smn\nf05960/Df(3L)Smn\nX7 heterozygotes die between the 2nd and 3rd instar larval stages, while ∼60% of Smn\nf01109/Df(3L)Smn\nX7 are viable. Therefore, using lethality as a criterion, all three alleles behave as loss-of-function mutations with Smn\nf01109 displaying the weakest phenotype of the three. No obvious maternal or paternal effect is observed for the different alleles. m: maternal contribution, p: paternal contribution. WT is wild-type (Canton-S). At least 100 individuals were examined for each genotype.Constructing RNAi-based hypomorphic Smn allelesSince the clinical severity of SMA correlates with the amount of SMN expression, we sought to better model the disease by generating a set of Smn alleles with varying degrees of SMN activity using RNAi. A GAL4-inducible vector was used to produce three different double-stranded RNAi transgenic constructs targeted against the full-length SMN protein (FL) as well as the amino-terminal (N) (the entire 5′ portion of the protein up to and including the Tudor domain) and carboxy-terminal (C) (the 3′ portion of the protein after, but not including, the Tudor domain) SMN regions (Fig 2A).Ten independent transgenic strains for each type of construct (C, N and FL) were generated and examined for their effects on lethality when SMN activity was reduced or eliminated using either tubulinGAL4 or actinGAL4, two ubiquitous GAL4 drivers. It was difficult to differentiate between the lethal phases of many strains in the tubulinGAL4 background, presumably due to its higher levels of expression. Instead, we were able to use the timing of lethality in the presence of actinGAL4 to choose three lines ([UAS-Smn-RNAi]N4 (N4), [UAS-Smn-RNAi]C24 (C24) and [UAS-Smn-RNAi]FL26B (FL26B)) that define a set of alleles representing the broadest range of detectable lethality for further analysis (Figure 3A).10.1371/journal.pone.0003209.g003Figure 3Lethality strongly associates with loss of Smn function in muscle.Survival rates of animals expressing the N4, C24 and FL26B transgenic UAS-Smn-RNAi constructs under the control of the actinGAL4 (A), how24BGAL4 (B), and elavGAL4 (C) drivers were measured at the following developmental stages: embryo (day 0), 1st instar larva (day 2), 3rd instar larva (day 5), early pupa (day 7), late pupa (day 9), 2-day old adult (day 12). Each experiment was performed in triplicate. The empty pWIZ RNAi vector served as a control. The survival rates of animals were calculated and subtracted from control values. The N4, C24 and FL26B transgenic animals displayed graded viability among the drivers tested. Ubiquitous SMN knockdown (A) leads to pupal lethality. Muscle-specific SMN knockdown (B) leads to late pupal lethality only in animals harboring the stronger alleles (N4 and C24), whereas greater than 90% of FL26B individuals survive to adulthood. In contrast, reduction of SMN in neurons using N4 and C24 (C) causes only very mild lethality (7%) when compared to control animals. (D) Western blots using an anti-SMN polyclonal antibody show reduction of SMN protein in 3rd instar larvae for all three UAS-Smn-RNAi transgenic strains in combination with the ubiquitous actinGAL4 driver. The top panel shows a graded effect on SMN protein levels by the three constructs consistent with their effects on lethality. The bottom panel shows anti-α tubulin levels, which served as loading controls.Of all strains generated, N4 displayed the most severe phenotype, causing mortality at the early pupal stage. C24 was less severe and results in lethality at a later pupal stage than N4, while FL26B was semi-viable and was therefore the weakest allele of the three (Figure 3A). Under the control of the tubulinGAL4 driver, N4 caused a similar phenotype to those observed for the Smn\n73Ao and Smn\nf05960 mutations, suggesting that N4 is a strong hypomorphic Smn allele (data not shown). The efficiency of RNAi in the N4 and C24 strains precluded us from testing whether ectopic SMN expression could rescue the RNAi-induced lethality. However, we do note that the fully penetrant pupal lethality induced by the expression of tubulinGAL4-directed FL26B is completely rescued by the addition of the UAS-FLAG-Smn construct to this genetic background (data not shown).Consistent with these results, examination of protein derived from 3rd instar larvae from the above strains in the presence of the actinGAL4 driver revealed significant reductions in SMN expression levels (Figure 3D), further suggesting the observed lethality is the direct result of SMN protein attenuation. Though the three strains did not display apparent differences in the degree of reduction of SMN under these conditions, the genetic results with respect to viability and subsequent experiments investigating NMJ morphology (see below) strongly suggest these RNAi-induced Smn strains result in varying degrees of SMN activity and therefore, alleles of different strengths. Importantly, these reagents provide important genetic tools that will allow us to examine the requirement of SMN in muscle and neurons.Loss of Smn causes neuromuscular junction defectsSMA patients experience motor neuron degeneration and muscle atrophy [1], [4]. Consistent with this, previous work has shown that a loss of Smn function results in defects at the Drosophila NMJ [15]. To confirm and extend these results, we examined the NMJ phenotype observed in various Smn genetic backgrounds by quantitatively assessing the morphology of the NMJ through examination of synaptic bouton numbers between muscles 6 and 7 of the 3rd instar larval NMJs. These boutons are visualized by using antibodies against the Synaptotagmin (SYT) (pre-synaptic) and DLG (post-synaptic) proteins, respectively (Figure 4A–G) (Materials and Methods\n[29], [31], [32]). The following Smn genotypes, which were capable of reaching the 3rd instar larval stages (Smn\n73Ao/Smn\nf01109, Smn\nf05960/Smn\nf01109 and Smn\nf01109/Smn\nf01109) and therefore amenable to dissection, were examined.10.1371/journal.pone.0003209.g004Figure 4\nDrosophila Smn mutations elicit neuromuscular junction (NMJ) defects.(A–F) The morphology of the NMJ, as judged by bouton numbers, between muscles 6 and 7 in the A2 segment was observed in different genetic backgrounds using the pre-synaptic (Synaptotagmin) and post-synaptic (Discs large) markers, shown in green and red, respectively. The following genotypes were examined: (A) wild-type (Canton-S), (B) Smn\nf05960/Smn\nf01109 (C) Smn\nf01109/Smn\nf01109, (D) Smn\n73Ao/Smn\nf01109. Of these combinations, Smn\n73Ao/Smn\nf01109 displayed the most robust NMJ defect. These defects are partially rescued by either (E) neuron-specific expression (elavGAL4) or (F) muscle-specific expression (how24BGAL4) of a UAS-FLAG-Smn transgene. (G) More complete rescue was achieved when this transgene was expressed using both drivers simultaneously. Bouton numbers were normalized to the ratio of the muscle area. Scale bars represent 20 µm. (H) Diagram of bouton numbers for genotypes from (A–F), normalized for muscle area. * P<0.05 was determined by the ANOVA multiple comparisons test. For each genotype at least 15 animals were examined.The most severe reduction in NMJ bouton numbers was observed in a Smn\n73Ao/Smn\nf01109 genetic background (Figures 4A–D and F). The semi-viable Smn\nf01109 mutation displayed a moderate reduction in NMJ bouton numbers, consistent with its weakly hypomorphic nature (Figure 4C). Surprisingly, the strong loss of function Smn\nf05960 mutation, though homozygous lethal, failed to exhibit a detectable change in NMJ bouton numbers in an Smn\n73Ao background. However, an increase in pre-synaptic ghost bouton numbers [33], [34] (where pre-synaptic SYT was not accompanied with post-synaptic DLG) was observed in these individuals (Figure S3), indicating that the Smn\nf05960 allele does, indeed, disrupt NMJ morphology. The NMJ phenotype associated with Smn\n73Ao/Smn\nf01109 individuals was rescued partially by neuronal or muscle-directed expression of a UAS-FLAG-Smn transgene (Figure 4E–G), suggesting that SMN expression in either tissue is sufficient to restore, at least partially, NMJ morphology.Loss of Smn function in muscles causes lethalityThough it is clear that global reduction of SMN function elicits a larval lethal phenotype (Figure 2B), the relative requirement of SMN in muscle versus neuron remains unresolved. We sought to address this question directly through use of our inducible Smn RNAi strains (N4, C24 and FL26B), which can be expressed using tissue-specific GAL4 drivers. Therefore, we chose to reduce SMN expression in neuronal and muscle lineages using the pan-neuronal elavGAL4\n[35] and pan-muscle how24BGAL4 drivers, respectively (how24BGAL4 is a mesodermal driver that expresses in all muscles, and in the remainder of the text we refer to it as a muscle driver) [36], [37].Reduction of SMN in either tissue causes lethality, however, loss of SMN expression in the muscle results in an earlier onset of lethality, which we consider to be a more severe phenotype (Figure 3B–C). In the strongest Smn RNAi allele, N4, muscle-specific SMN reduction results in 70% mortality (Figure 3B), while neuronal specific reduction results in 7% mortality (Figure 3C). As RNAi is less efficient in neurons, we added a GAL4-driven dicer construct to increase the efficacy of SMN reduction under these conditions [38]; this resulted in no obvious enhancement of lethality in all Smn RNAi and elavGAL4 backgrounds (data not shown). The GAL4 repressor GAL80 was expressed in neurons using the pan neuronal n-syb driver [39] to overcome the potential leakiness of the how24BGAL4 driver. Since the lethality observed for muscle specific reduction of SMN more closely resembles ubiquitous SMN reduction (compare Figure 3A and B), these indicate the requirement of SMN in the muscle (using how24BGAL4) is more important for viability than its requirement in the neurons.Muscle and neuronal expression is required for normal NMJ morphologySimilar to the tissue-dependent lethality experiments above, we sought to assess the impact SMN activity has on NMJ morphology using our UAS-Smn-RNAi strains, which can be expressed using tissue-specific GAL4 drivers.We selectively reduced SMN expression in neuron and muscle tissues by crossing the UAS-Smn-RNAi alleles to the elavGAL4 and how24BGAL4 drivers as they provide the earliest tissue specific expression and most robust lethal effect (Figure 3 and data not shown). Visualized by SYT (pre-synaptic) and DLG (post-synaptic) staining, NMJs of Smn RNAi animals containing either a muscle- or neuron-specific GAL4 driver revealed a reduction in the number of synaptic boutons compared to vector alone controls (Figure 5A–M). In the N4 strain, both neuron and muscle specific attenuation of SMN cause approximately 50% reduction in bouton numbers (Figure 5B, C, K–M), a reduction comparable to what is observed in Smn\n73Ao/Smn\nf01109 larvae (Figure 4D, H). Therefore, we conclude that the NMJ morphology is dependent upon both pre- and post-synaptic SMN activity.10.1371/journal.pone.0003209.g005Figure 5Muscle and neuron specific Smn RNAi knockdown causes NMJ defects.(A–I) Reduced SMN expression in the N4, C24 and FL26B UAS-Smn-RNAi transgenic constructs elicits graded effects on NMJ morphology using the ubiquitous actinGAL4 (A, D, G) as well as the tissue-specific how24BGAL4 (muscle) (B, E, H) and elavGAL4 (neuron) (C, F, I) drivers. Vector only (pWIZ) controls are shown (J, K, L). In these images the pre- and post-synaptic tissues are labeled with antibodies against Synaptotagmin (green) and Discs large (red), respectively. (M) Bouton counts for the NMJs from the genotypes shown in (A–L) were normalized for muscle area and subtracted from vector only controls. For each genotype at least 15 animals were examined. * P<0.01 and **P<0.05 was determined by the ANOVA multiple comparisons test. Scale bars represent 15 µm.Previous studies demonstrated that mutations in Smn cause a decrease in staining for the post-synaptic neurotransmitter receptor subunit, GluRIIA [15]. To corroborate these results and to extend our characterization of the tissue-specific requirement of SMN at the NMJ, we examined the GluRIIA [40], [41], [42] expression pattern (See Materials and Methods) in the UAS-Smn-RNAi backgrounds. We found a consistent and significant quantitative reduction in synaptic GluRIIA levels when Smn expression was decreased using either neuron- (elavGAL4) or muscle-specific (mhcGAL4) drivers. GAL4-only controls had no significant effect on GluRIIA staining intensity. Consistent with the trend observed for the severity of the lethal phenotype, the strongest Smn RNAi alleles caused the greatest reduction in GluRIIA expression levels, suggesting that GluRIIA levels are sensitive to the dose of functional SMN protein and thus, would be a useful phenotypic metric in which to validate potential modifiers of the Smn NMJ phenotype.Our analysis indicates that normal NMJ morphology requires SMN activity in both muscle and neurons. However, it appears that loss of SMN activity in the muscle causes a more severe lethal phenotype (Figure 3B), a conclusion that is consistent with the finding that the SMN protein is concentrated in the post-synaptic regions in muscle (Figure 1A–D).Identification of genetic modifiers of Smn\nTo gain insights into the genetic circuitry capable of modulating SMN activity in vivo, we employed a genetic approach to screen for genes that affect Smn-dependent processes using the Exelixis collection of transposon-induced mutations [25], [26]. The benefits of using the collection in a genetic screen have been previously described [24]. Notably, the collection covers approximately 50% of the genome and harbors both gain- as well as loss-of-function mutations when exposed to GAL4 due to the presence of UAS sequences within the insertional transposons [25], [26]. While the molecular coordinates of each insertion site is known, gene assignments are sometimes ambiguous, as the modifying transposon may have inserted between two genes.The screen was carried out in two stages to identify both enhancers and suppressors of Smn-associated lethality (Figure 6). The strong correlation observed between the degree of lethality and NMJ phenotypes using the Smn RNAi lines suggested the use of lethality as a screening parameter would be successful in identifying components of the SMN genetic network that might also affect the NMJ. Both phases of the screen utilized the Smn73Ao allele, which gives a robust NMJ defect, and importantly, contains a point mutation in the YG box (Figure 2A), which is the location of a documented human SMN1 mutation [3].10.1371/journal.pone.0003209.g006Figure 6Schematic representation of the Smn modifier screen.Depicted are the crosses performed to identify enhancers and suppressors of Smn-associated lethality. In the first stage of the screen, designed to identify Smn enhancers, Smn\n73Ao\ntubulinGAL4 e/TM6B virgin females were mated to males from Exelixis collection strains. In this stage, the entire Exelixis collection, which affects approximately 50% of the Drosophila genome, was tested. In the F1 generation, mutations that resulted in synthetic lethality or reduced viability in trans with the Smn\n73Ao\ntubulinGAL4 e chromosome were defined as enhancers. In the second stage of the screen, males from F1 crosses that failed to show enhancement (P[Exelixis]/+; Smn\n73Ao\ntubulinGAL4 e/TM6B) were mated to Smn\n73Ao\ne/TM1, Mé virgin females to identify mutations that suppressed the Smn\n73Ao\ntubulinGAL4 e/Smn\n73Ao, e lethal phenotype. We performed the F2 suppressor screen with Exelixis mutations on first and second chromosomes as testing third chromosome mutations would require placing these mutations in cis with Smn. Additional assays were employed to eliminate false positives (See Materials and Methods). Seventeen enhancers and ten suppressors met these criteria. All 27 modifiers were subsequently examined for their ability to modify the Smn NMJ phenotype by GluRIIA staining (Figures S5 and S6).The first stage was an F1 screen designed to identify insertions that produced synthetic lethality or semi-lethality (Materials and Methods) in an Smn heterozygous background, which will hereafter be referred to as enhancers. Using this criterion, we screened the entire Exelixis collection and identified 17 insertions that result in Smn\n73Ao/+ lethality (Figure 7).10.1371/journal.pone.0003209.g007Figure 7Modifiers of Smn phenotypes.Listed are the insertions that enhance (top) or suppress (bottom) Smn\n73Ao-dependent lethality. Due to the site of transposon insertion, unambiguous gene assignments were not possible in all instances (shaded). Strains whose designations begin with “d” or “f” contain GAL4 responsive elements (UAS), whereas strains beginning with “c” or “e” are not GAL4-inducible. Gene assignments were determined using FlyBase (http://www.flybase.org/). Human homologs were determined using NCBI BLAST, NCBI UniGene (NCBI) (http://www.ncbi.nlm.nih.gov/sites/entrezdbunigene) or ENSEMBL genome browser (http://www.ensembl.org). Annotated functions were determined based on FlyBase, NCBI Entrez Gene and SMART (http://smart.embl-heidelberg.de/). Modification of the NMJ morphology between muscles 6 and 7 in the A2 segment was assayed in the elavGAL4 pWIZ[UAS-Smn-RNAi]C24 background in trans with all identified modifiers using the pre-synaptic (Horseradish peroxidase) and post-synaptic (GluRIIA) markers (see Materials and Methods). In the three cases that did not show significant phenotypic alteration, additional pWIZ[UAS-Smn-RNAi]N13 allele was also used (see text). The degrees of change observed in GluRIIA staining were categorized as follows: +++, strong; ++, moderate; +, weak; N.E., No Effects.In the second stage of the screen we tested for the ability of mutations to suppress Smn-dependent larval lethality. This was accomplished using offspring from the F1 screen that failed to generate synthetic lethality. In this phase, we screened 7170 strains (as Smn\n73Ao is located on the third chromosome, we excluded third chromosome insertions) and identified ten suppressors of homozygous Smn\n73Ao lethality (Figure 7).NMJ analysis of Smn modifiersTo correlate modifier activity with the NMJ, we investigated whether all of the Smn modifiers (10 suppressors and 17 enhancers) could disrupt Smn RNAi-dependent NMJ defects, using synaptic GluRIIA staining as an assay to quantify the degree to which the Smn phenotype was modified by the interacting mutation. For this assay, we employed the C24 Smn RNAi line because it displays intermediate phenotypic strength. In all but two cases, the combination of the modifier insertion mutation induced a statistically significant change in the C24 GluRIIA phenotype (Figure 7 and Figure S5 and S6). Amongst the validated modifier insertions, the degree of enhancement or suppression varied depending on the locus; control crosses demonstrated that there were no significant Smn-independent changes in GluRIIA localization for the tested insertion lines. Three lines (f04448, d09801 & d00698) failed to modify C24 GluRIIA staining and were retested using a weaker Smn RNAi strain, N13 (Strain f04448 and d09801 enhanced, whereas d00698 showed no interaction (data not shown), highlighting the importance of the NMJ phenotype as a secondary screening tool (Figure 7)). Thus, the majority modifiers of the Smn\n73Ao lethal phenotype were confirmed by a second, independent assay. All but one of these insertions modified the Smn NMJ phenotype, validating the efficacy of the screen and suggesting that the screen may prove to be an effective tool in the identification of candidate genes that may be relevant to the SMA disease state.Neuronal overexpression of wishful thinking (wit) enhances Smn NMJ defectsTo validate further our approach, we sought to examine the relationship between wishful thinking (wit) and Smn in greater detail. wit was of particular interest because it has been previously implicated in NMJ function [27], [28] and thus could serve as a paradigm for validating the ability of the screen to identify bona fide Smn genetic modifiers.\nwit encodes a type II BMP receptor that functions as a retrograde signaling component in neurons [27], [28]. wit loss-of-function mutations cause NMJ defects, whereas wit gain-of-function causes no obvious NMJ morphological changes. As the wit allele identified as an Smn enhancer, wit\nd02492, is associated with a GAL4-responsive transposon, it seemed likely that it represented a gain-of-function mutation. Consistent with this notion, an independent UAS-wit transgene [27], [28] behaved in a similar fashion to wit\nd02492 under the conditions used in our screen (data not shown). In addition, we detected increased expression of WIT in wit\nd02492 animals containing tissue-specific GAL4 drivers (data not shown).Over-expression of WIT in neurons using the neuron-specific elavGAL4 driver in either an Smn\n73Ao or an Smn\nf01109 heterozygous background resulted in reduced NMJ bouton numbers relative to elavGAL4 Smn\n73Ao/+, elavGAL Smn\nf01109/+ and UAS-wit; elavGAL4 controls (Figure 8). This result suggests that the Smn-dependent NMJ phenotype is sensitive to elevated WIT levels.10.1371/journal.pone.0003209.g008Figure 8\nwit overexpression in neurons exacerbates Smn-dependent NMJ defects.A gain-of-function mutation of wishful thinking (wit), wit\nd02492, was identified as an enhancer in our screen. To further investigate the interaction between wit and Smn at the NMJ, we used the neuron-specific driver, elavGAL4 to express WIT in neurons. (A–F) The morphology of the NMJ, as judged by bouton numbers, between muscles 6 and 7 in the A2 segment was observed in different genetic backgrounds using the pre-synaptic (Synaptotagmin) and post-synaptic (Discs large) markers, shown in green and red, respectively. The following genotypes were examined: (A) elavGAL4/+, (B) elavGAL4, Smn\n73Ao/+, (C) elavGAL4, Smn\nf01109/+, (D) elavGAL4/UAS-wit2A, (E) elavGAL4, Smn\n73Ao/UAS-wit2A, (F) elavGAL4, Smn\nf01109/UAS-wit2A, (G) Bouton counts for genotypes from (A–F and wild-type). Consistent with previous reports, neural induced expression of the UAS-wit2A transgene had no obvious effect on NMJ bouton number. A synergistic effect was observed upon the addition of a single Smn allele (Smn\n73Ao or Smn\nf01109) to this background, leading to a reduction of NMJ bouton numbers. The phenotype was more severe in the Smn\nf01109 background. Smn\nf01109 showed an approximate 50% reduction in bouton numbers while Smn\n73Ao reduced the bouton count by 20%. elavGAL4, Smn\n73Ao/+ (B) and elavGAL4, Smn\nf01109/+ (C) individuals display no significant reduction in NMJ bouton numbers compared to wild-type (G). Bouton counts were determined as above. Error bars are s.e.m.; * P<0.02 was determined by the ANOVA multiple comparisons test to wild-type and all controls. n was 15–20 animals for each genotype. Bouton numbers for each genotype were normalized to the ratio of muscle areas. Scale bars represent 20 µm.A Mad mutation enhances the Smn NMJ phenotypeGiven the involvement of wit at the NMJ and its interaction with Smn, we hypothesized that an Smn heterozygous background leads to an increase in sensitivity to the dosage of BMP during NMJ development. Thus, under conditions of elevated levels of WIT in Smn heterozygotes, it is possible that normal BMP signaling at the NMJ is altered, perhaps due to titration of the BMP ligand, thereby resulting in NMJ defects. If this hypothesis is correct, mutations of the BMP components downstream of wit should also enhance the Smn NMJ phenotype. Therefore, we tested whether Mothers against dpp (Mad) and Smn interaction at the NMJ. Mad encodes the Drosophila homolog of R-Smad, a downstream effector of the pathway [34], [43], [44]. Pathway activation leads to phosphorylation of MAD (pMAD), and its subsequent translocation to the nucleus where it regulates gene expression [34], [43], [44]. To examine the consequences of Smn/Mad interaction at the NMJ, we used the hypomorphic Mad\n12 allele [34] in combination with multiple Smn alleles to monitor the phenotypic effects at the NMJ. The moderate reduction in number of NMJ boutons caused by the hypomorphic Mad\n12 allele (Figure 9D and G) is clearly exacerbated by mutations in Smn (Figure 9E–G). These results suggest that perturbations in BMP signaling are able to modify Smn-dependent phenotypes at the larval NMJ.10.1371/journal.pone.0003209.g009Figure 9Loss of mad function enhances Smn NMJ defects.(A–F) The morphology of the NMJ, as judged by bouton numbers, between muscles 6 and 7 in the A2 segment was observed in different genetic backgrounds using the pre-synaptic (Synaptotagmin) and post-synaptic (Discs large) markers, shown in green and red, respectively. The following genotypes were examined: (A) wild-type, (B) Smn\n73Ao/+, (C) Smn\nf01109/+, (D) mad\n12/+, (E) Smn\n73Ao/mad\n12 and (F) Smn\nf01109/mad\n12. (G) Bouton counts for genotypes in (A–F). Introduction of mad\n12 into either a Smn\n73Ao/+ or a Smn\nf01109/+ background dominantly reduces the Smn-dependent NMJ bouton count. Error bars are s.e.m.; *P<0.02 was determined by the ANOVA multiple comparisons test to wild-type and all controls. n was 15–20 animals for each genotype. Bouton numbers for each genotype were normalized to the ratio of muscle areas. Scale bars represent 20 µm.SMN activity affects BMP signalingTo further validate the link between SMN and the BMP signaling pathway we examined the effect of reduced SMN levels on pMAD expression. Though Mad is required for retrograde signaling in neurons at the NMJ [34], [45], a lack of detectable pMAD staining at the NMJ precluded the use of the NMJ as a means to assess whether SMN can affect its expression. Instead, we examined the pMAD expression pattern adjacent to the anterior-posterior compartment boundary of 3rd instar larval wing discs [46] (Figure 10) using engrailedGAL4 and vestigalGAL4 directed expression of the N4 RNAi transgene (Figure 10 and Figure S4 respectively). Regions in which SMN levels are reduced display attenuated pMAD staining (Figure 10C–E). Moreover, adult wing abnormalities occur in regions of reduced SMN expression, including thicker wing veins and shorter posterior cross-veins (Figure 10F). These phenotypes are similar to phenotypes elicited by mutations in other BMP pathway components such as thickveins (tkv) and glass bottom boat (gbb) [45], [47], [48]. Thus, BMP signaling in the wing appears to be affected by loss of SMN activity through the regulation of activated Mad, corroborating the link between Smn and the BMP signaling pathway.10.1371/journal.pone.0003209.g010Figure 10Smn knockdown reduces pMAD signals.(A–B) Wild-type wing discs from 3rd instar larvae were stained with antibodies against SMN (red) (A) and phosphorylated MAD (pMAD) (green) (B). (C–D) 3rd instar wing discs of engrailedGAL4, pWIZ[UAS-Smn-RNAi]N4 animals are stained with antibodies against SMN (red) (C) and pMAD (green) (D). (E) Merge of (C) and (D). pMAD staining is reduced in the posterior region of the wing disc where SMN expression is decreased (yellow line). (F) A wing from an engrailedGAL4, pWIZ[UAS-Smn-RNAi]N4 transgenic adult exhibits defects in the posterior crossvein regions and the distal portions of wing veins L4 and L5 (arrow). Scale bars represent 40 µm.A Dad loss of function allele is capable of rescuing Smn NMJ defectsWe extended these observations by probing the relationship between the BMP pathway antagonist, Daughters against dpp (Dad), and Smn. Dad encodes the Drosophila homolog of mammalian anti-Smad and acts as a Mad antagonist [44], [49], [50]. Since Dad mutants exhibit pre-synaptic overgrowth [49], we tested whether the Dad\n271-68 null mutation could rescue the Smn NMJ phenotype. Consistent with previous reports [49], 3rd instar larvae homozygous for Dad\n271-68 display more dispersed SYT expression at the NMJ than control larvae (Figure 11C). However, in contrast to previous studies, we found the total bouton number, as determined by DLG post-synaptic staining, was only slightly reduced. Importantly, the Smn\n73Ao/Smn\nf01109 NMJ phenotype was suppressed by the introduction of Dad\n271-68 (Figure 11B, D, E), providing genetic evidence that a third element of the BMP pathway interacts with Smn. It appears that elevating BMP activity through a complete loss of Dad function suppresses the effects of Smn mutations on the NMJ (Figure 11D, E). A prediction of this model is that pharmacological reagents that increase BMP signaling may ameliorate Smn-associated NMJ defects, thereby identifying a set of targets of potential therapeutic value.10.1371/journal.pone.0003209.g011Figure 11A dad null allele rescues Smn NMJ defects.(A–D) The morphology of the NMJ, as judged by bouton numbers, between muscles 6 and 7 in the A2 segment was observed in different genetic backgrounds using the pre-synaptic (Synaptotagmin) and post-synaptic (Discs large) markers, shown in green and red, respectively. The following genotypes were examined: (A) wild-type (B), Smn\n73Ao/Smn\nf01109, (C) dad\n271-68 homozygotes and (D) Smn\n73Ao\ndad\n271-68/Smn\nf01109\ndad\n271-68. (E) Bouton counts for genotypes in (A–D). Smn\n73Ao/Smn\nf01109 individuals display strongly reduced NMJ bouton numbers while dad\n271-68 homozygotes have a greater than two-fold of bouton numbers relative to the Smn\n73Ao/Smn\nf01109 animals. The Smn\n73Ao\ndad\n271-68/Smn\nf01109\ndad\n271-68 double mutants behave like dad\n271-68 homozygotes. Error bars are s.e.m.; n is 15–20 animals for wild-type and Smn\n73Ao/Smn\nf01109. n is 30 for dad\n271-68/dad\n271-68 and Smn\n73Ao, dad\n271-68/Smn\nf01109, dad\n271-68. *Π<0.002 by the ANOVA multiple comparisons test. Bouton numbers for each genotype were normalized to the ratio of muscle area. Scale bars represent 15 µm.Discussion\nSMN1 is the determining gene for Spinal Muscular Atrophy (SMA) [5], a devastating neurodegenerative disease in humans with no currently available FDA-approved drug treatment. Though the general biochemical function of SMN in snRNP assembly has been well documented [51], [52], [53], much remains to be learned about its action at the NMJ and the genetic circuitry that is capable of affecting SMN activity. Specifically, it remains unclear whether the NMJ pathology in SMA is due to the ubiquitous loss of SMN function or whether SMN has a unique role at the NMJ. Here, we have utilized Drosophila to investigate the tissue specificity of Smn and to identify genes that interact with Smn. These genes, apart from their intrinsic value in providing insight into the role of SMN at the NMJ, may also define novel therapeutic targets.Previous studies based primarily on the analysis of the Smn\n73Ao allele demonstrated that reduced Smn activity causes lethality and NMJ morphological defects [15]. We corroborated these observations through the examination of several extant and novel Smn mutations of varied severities, including several GAL4-inducible Smn RNAi alleles generated for this study. These hypomorphic strains reduce SMN expression levels to different degrees in a manner formally analogous to decreased SMN levels observed in SMA patients. Additionally, these strains may model the dosage-dependent nature of SMA [1], [2] as the developmental arrest associated with these animals correlates with the extent of morphological abnormalities observed at the NMJ.Our examination of Smn NMJ structure in Drosophila using pre- and post-synaptic markers, SYT and DLG, respectively, revealed significant losses of synaptic bouton numbers in multiple Smn backgrounds (Figure 4). Moreover, in these backgrounds, we also detected reduced post-synaptic GluRIIA expression (data not shown), consistent with previous analyses of the Smn\n73Ao NMJ [15]. Together, these results suggest that loss of SMN function in Drosophila causes aberrant neuromuscular synaptic structure, mimicking the pathology of SMA [1], [54]. In addition, these structural abnormalities are consistent with the altered electrophysiological profile previously observed in Drosophila Smn\n73Ao animals [15]. It should be noted that other glutamate receptor subunits display altered transcriptional profiles in a Smn\n73Ao background; specifically the GluRIIA and GluRIIB transcript levels were decreased while GluRIIC levels were increased [55]. Therefore, combining genetic and morphological analyses of pathological changes in synaptic structure with future electrophysiological studies will be necessary to understand more thoroughly the synaptic consequences of SMN loss in SMA.A longstanding question in the pathology of SMA is the relative neuronal and muscle contribution of SMN function. The RNAi strains allowed us to reduce SMN function in a tissue-specific fashion and therefore, address this issue directly. We find that SMN is required in both neurons and muscle for normal NMJ morphology as GAL4-inducible RNAi reduction of SMN in neurons and muscle both show a decrease in NMJ bouton numbers (Figure 5) and GluRIIA staining (Figures S5 and S6 and data not shown). In addition, expression of SMN in either tissue is sufficient to partially rescue NMJ defects associated with loss of Smn function (Figure 4E–G). These results are consistent with previous reports in Drosophila, zebrafish and mouse [15], [16], [54], [56], [57], [58], [59] that indicate an interdependence of neuron and muscle SMN activity.In contrast to a requirement for Smn in both muscle and neurons at the NMJ, we demonstrated that muscle specific reduction of Smn causes a more severe lethal phenotype (Figure 3B–C). We do not know the cause of the lethality. It is possible that the earlier onset of lethality observed for the how24BGAL4 reduction of SMN may result from the leakiness of the driver or loss of SMN activity in dividing cells (the elavGAL4 driver expresses predominantly in post-mitotic cells). However, our results raise the possibility that the organism is more vulnerable to SMN reduction in the muscle. This is also consistent with the post-synaptic concentration of SMN at the NMJ (Figure 1). The functional relevance of these observations remains to be determined; however, a previous report has suggested that Smn may have a specific function in the Drosophila adult skeletal muscle where SMN is expressed in the sarcomere and was shown to bind to α-actinin [18]. Together, these data provide plausible explanations why muscle may be rendered more susceptible to loss of Smn function.Current therapeutic strategies for treatment of SMA are based on the dosage dependent nature of the disease, focusing on drugs that increase SMN2 transcription and splicing efficiency [60], [61]. Though these strategies may ultimately prove successful in treating SMA, complementary therapies may allow for the delivery of a combination of drugs as this has been shown to be successful in alleviating the symptoms of other diseases, such as AIDS [62]. Hence, the identification and, ultimately, the manipulation of genetic elements that affect SMN activity may be necessary to treat SMA effectively. Though previous biochemical studies provide valuable and fundamental knowledge of SMN function, our current understanding of SMN has been limited mainly to its binding partners and a few genetic modulators [12], [19]. Thus, we performed a genome-wide genetic screen in Drosophila to identify novel components of the Smn genetic circuitry to broaden our knowledge of its function and to seek potentially novel therapeutic approaches beyond the augmentation of SMN2 expression.Since we observed that the severity/onset of Smn-dependent mortality (Figure 3) corresponds to the degree of NMJ defects (Figure 5), we reasoned the identification of enhancers and suppressors of Smn\n73Ao-dependent lethality would be likely to yield genes that also function at the NMJ. Our genetic screen using an allele (Smn\n73Ao) that encodes a point mutation seen in SMA patients [3] resulted in the identification of twenty-seven modifiers of Smn lethality. Though we recognize the genetic circuitry in Drosophila may differ from that which exists in humans, we expect there to be substantial overlap given the conservation of gene function across species.Despite the essential role of SMN in snRNP assembly [12], an unexpected result of the screen was that none of the modifying insertions for which unambiguous gene assignments were made appear to function in RNA processing. Consistent with this notion, direct attempts to identify genetic relationships between SMN and known components of the SMN multimeric complex, including deficiencies that uncover the Drosophila Gemin homologs, did not affect the Smn\n73Ao heterozygous phenotype (data not shown). One possible explanation is that removal of additional components of the SMN complex may not enhance Smn-related phenotypes since SMN activity is critical for the initial steps in SMN complex assembly. Hence, altering the activity of “downstream” or directly-interacting partners of the SMN in the SMN complex may not affect Smn-related phenotypes.Though none of the unambiguously identified modifier genes have an obvious role in snRNP assembly; we did recover genes (wishful thinking, fmr1 and cutup) that have been shown previously to function at the NMJ [27], [28], [63], [64]. Moreover, the majority of the remaining genes, which had no previously known NMJ function, also modified Smn NMJ phenotypes. Thus our genetic approach was efficient in identifying genes related to Smn NMJ function. This suggests that a similar approach utilizing a hypomorphic Smn allele (e.g. UAS-Smn-RNAi) that more closely approximates the dosage dependent nature of the human disease condition may identify additional members of the Smn genetic circuitry.An analysis of the interacting loci according to molecular functions reveals an assortment of functional categories including cytoskeleton interaction proteins (moe and ctp), transcription factors (net) and metabolic enzymes (CG17323 and CG10561). Identified interactors also include members of several signal transduction pathways (e.g. BMP (wit), FGF (btl) and Nuclear Hormone Receptor (Eip75B)), raising the possibility that these evolutionarily conserved signaling pathways integrate with SMN or targets of SMN function(s). Though more detailed analyses of the nature of the links (synergistic or parallel) between these pathways and SMN are necessary, we provide strong evidence supporting a connection between BMP signaling and Smn at NMJ by testing additional upstream and downstream elements of this pathway. Our molecular genetic analysis clearly indicates that SMN influences BMP activity. It remains to be determined whether SMN acts in the muscle to influence retrograde BMP signaling through the WIT receptor, for example by regulating the activity of the WIT ligand (GBB). It is also possible that SMN functions cell-autonomously in the neurons to affect the activity of MAD or its antagonist, DAD. As the BMP signaling pathway has been implicated in other neurodegenerative diseases, including Duchenne Dystrophy and Marfan Syndrome [65], it is probable that BMP signaling also plays a role in the pathology of SMA in humans.Similar to what is observed in SMA, our results confirm the susceptibility of the Drosophila NMJ to lower levels of SMN, and our screen has also identified several genes that modify Smn NMJ phenotypes. In other recent studies, micro-array based approaches analyzed the effect of reduced Smn levels on tissue-specific gene expression at a genome-wide level [52], [55]. They identified genes whose splicing are susceptible to reduced SMN function [52] and genes involved in general metabolic processes [55]. These screens are clearly a valuable means to assess the housekeeping function of Smn. However, unlike the genes recovered from our screen, most of which affect NMJ structure, it remains to be determined whether the genes identified through transcriptional profiling are involved in the development and/or maintenance of the NMJ. Thus, our genetic approach has uncovered elements, revealing a potential NMJ-specific role for Smn.In this study, we have used Drosophila genetics to broaden our understanding of Smn at the neuromuscular junction and probe the genetic circuitry of Smn, illustrating the utility of a genetic approach in the identification of novel genes that impact Smn activity. Given the high degree of genomic conservation, use of model systems such as Drosophila will, in our view, lead to a more thorough understanding of SMA pathology and point to potential therapeutic strategies.Materials and Methods\nDrosophila stocksAll stocks were maintained on standard cornmeal/yeast/molasses/agar medium at 25°C. The Smn\n73Ao, P{EPgy2}EY14384, wit\nA12 and wit\nB11 alleles were obtained from the Bloomington Drosophila Stock Center (Bloomington, IN). The Smn\nf05960 and Smn\nf01109 alleles are from the Exelixis collection at Harvard Medical School. Dad\n271-68 and Mad\n12 were gifts from Graeme Davis. P[UAS-wit2A] transgenic animals were gifts from Hermann Aberle. To generate the P[UAS–FLAG-Smn] strains, a full-length Smn cDNA was cloned into the amino-terminal FLAG-tagged vector, FLAG-pUAST and introduced into w\n1118 animals. Several independent strains were isolated and subsequently tested for their ability to rescue Smn-dependent lethality in a homozygous Smn\n73Ao\ne background. The majority of homozygous Smn\n73Ao\ne mutants die between 2nd and 3rd larval stages, and less than 10% reach the 3rd instar larval stage. Expression of this construct using the ubiquitous actinGAL4 and tubulinGAL4 drivers partially suppressed this lethality, as 50% of the animals survived to the late pupal stage and 20% survived to the adult stage. The null allele of Smn, Df(3L)Smn\nX7, was generated by imprecise excision of the P{EPgy2}EY14384. 1,626 excision events were isolated and 17 failed to complement Smn\n73Ao, including Df(3L)Smn\nX7. Subsequent sequence analysis of Df(3L)Smn\nX7 determined the excision event removed almost the entire SMN transcript without affecting nearby loci (93 bp upstream of the transcription start site through all but the final 44 bp of the 3′ UTR). To generate the Smn RNAi constructs, three different portions of the Smn cDNA were cloned into the pWIZ vector: the entire cDNA (FL constructs), the amino-terminal portion up to and including the entire Tudor domain (N constructs) and the carboxy-terminal portion after, but not including, the Tudor domain (C constructs) (Figure 2A). These constructs were then introduced into w\n1118 embryos by germ-line transformation according to standard procedures (by the CBRC fly core facility, Massachusetts General Hospital). Multiple independent insertions were obtained for each construct, including the pWIZ[UAS-Smn-RNAi]N4, pWIZ[UAS-Smn-RNAi]C24 and pWIZ[UAS-Smn-RNAi]FL26B transgenic strains that were used for the analyses conducted in this study. Detailed primer sets and methods used for sequencing Smn\nf05960 and Smn\nf01109 alleles are available upon request.Exelixis screenThe screen we performed combined elements from standard F1 and F2 screens. This “combination screen” was identical to a standard F2 screen with the exception that the crosses were designed to identify synthetic lethal interactions with Smn in the F1. In this screen we utilized the Smn\n73Ao allele in cis with a ubiquitously expressed tubulinGAL4 driver (Lee and Luo, 1999). Initially, Smn\n73Ao\ntubulinGAL4 e/TM6B virgin females were crossed to the entire Exelixis mutant collection to identify insertions that elicit F1 synthetic lethality or reduced viability. From these results, we arbitrarily defined a strain to be a candidate enhancer as one that displayed a viability of less than 30%. In the second generation, F1 males from strains that failed to elicit synthetic lethality were crossed to Smn\n73Ao\ne/TM1, Mé virgins to test for their ability to suppress homozygous Smn\n73Ao larval lethality. In the F2 screen, candidate suppressors were identified by the presence of individuals bearing the marker ebony (e), which is visible in both pupae and adults.Secondary genetic assaysWe used several secondary genetic assays to evaluate candidate modifiers. Initially, all candidate enhancers were crossed to tubulinGAL4 e alone, to determine whether the observed effects on viability were Smn-dependent.Resultant strains and all candidate suppressors were then crossed to stocks containing additional combinations of Smn alleles and GAL4 drivers to observe their effects on viability. These stocks included both Smn\nf05960 and Smn\n73Ao alone and in combination with the tubulinGAL4 or actinGAL4 drivers. The actinGAL4 driver alone was also tested. Twenty-seven strains modified Smn-dependent lethality. Of these, seventeen were classified as enhancers and ten as suppressors.NMJ analysis of 27 Smn interactorsStrains containing all twenty-seven interactors in combination with the pWIZ[UAS-Smn-RNAi]C24 were generated. These were tested for their ability to modify NMJ phenotype associated with elavGAL4 (neuronal) directed expression of pWIZ[UAS-Smn-RNAi]C24 and pWIZ[UAS-Smn-RNAi]N13. GluRIIA morphometric analyses were performed as described previously [66].Lethality assaysFertilized eggs were collected on apple juice agar plates. Before collection, adults were allowed to lay for 2 hours. All the F0 strains were balanced by TM6B, Dfd:YFP, Tb or CyO Dfd:YFP, and the non-YFP embryos were collected using a fluorescence dissection scope (Zeiss). Fertilized embryos were then place onto fresh apple juice plates containing yeast paste. Each plate contained 20–25 embryos to avoid over-crowding. The animals were allowed to grow into different developmental stages in controlled temperature (25 °C) and their survival was determined by visual inspection.Immunohistochemistry and microscopyPrimary antibodies were used at the following dilutions: monoclonal anti-DLG (1∶500) (Developmental Studies Hybridoma Bank), polyclonal anti-Synaptotagmin (1∶1000) (a gift from Hugo Bellen), polyclonal anti-SMN (1∶250, NMJ staining), monoclonal anti-SMN (1∶500, wing disc), polyclonal anti-pMAD (1∶250) (a gift from Carl-Henrik Heldin). Anti-SMN monoclonal and polyclonal antibodies were generated by immunizing animals with purified full-length SMN protein with a 6×His-tag fused to its carboxy-terminus (Cocalico Biologicals, Inc.). Texas-red conjugated anti-HRP (1∶250), FITC- (1∶40) and Cy5- (1∶40) conjugated anti-rabbit and anti-mouse secondary antibodies were purchased from Jackson Immunoresearch Laboratories. For the NMJ analyses, 3rd instar larvae were dissected and fixed for 5 minutes in Bouin's fixative. Imaginal disc dissections were performed on 3rd instar larvae in phosphate-buffered saline (PBS). Discs were kept on ice until fixation in 3% paraformaldehyde in PBS. Stained specimens were mounted in FluoroGuard Antifade Reagent (Bio-Rad), and images were obtained with a Zeiss LSM510 confocal microscope. Bouton numbers were counted based on the Discs large and Synaptotagmin staining in the A2 segment between muscles 6 and 7. The ratio of muscle area for the various genotypes was normalized to wild-type. GluRIIA morphometric analyses were performed as described previously [66].Supporting InformationFigure S1\nSpecificity of the anti-SMN antibodies. (A–C) Wing discs from 3rd instar larvae overexpressing the UAS-FLAG-Smn transgenic rescue construct using the vestigalGAL driver were stained with antibodies against the FLAG peptide (green) (A) and SMN (red) (B). (C) Merge of (A) and (B) showing the overlapping expression of SMN and FLAG within the vestigal expression domain. (D) Wild-type and (E) vestigalGAL4, pWIZ[UAS-Smn-RNAi]N4 3rd instar wing discs were stained with antibodies against SMN (green). (F) Western blots of a serial dilution of S2 cell extracts (1: 20 µg, 2: 40 µg, 3: 60 µg, 4: 80 µg total protein) using the polyclonal (left) and monoclonal (right) antiserum against SMN recognize a single band of approximately 28 kD in size.(4.46 MB TIF)Click here for additional data file.Figure S2\nSMN post-synaptic staining is abolished by muscle specific SMN knockdown. (A–F) The morphology of the NMJ between muscles 6 and 7 in the A2 segment was observed in different genetic backgrounds using antibodies against SMN (green) and the post-synaptic marker, Discs large (red). (A–C) Wild-type: anti-DLG (A), anti-SMN (B) and (C) merge of (A) and (B). (D–F) Transgenic animals containing how24BGAL4 and pWIZ[UAS-Smn-RNAi]N4: anti-DLG (D), anti-SMN (E) and (F) merge of (D) and (E). In this background, SMN staining is reduced (E).(2.42 MB TIF)Click here for additional data file.Figure S3\nPre-synaptic ghost bouton counts are elevated in \nSmn\n animals. The morphology of the NMJ between muscles 6 and 7 in the A2 segment was observed in different Smn backgrounds using the pre-synaptic (Synaptotagmin) and post-synaptic (Discs large) markers. Ghost bouton counts were determined by assessing the numbers of boutons that stained positive for Synaptotagmin and failed to stain for Discs large. All combinations examined (Smn\n73Ao/Smn\nf01109, Smn\nf05960/Smn\nf01109 and Smn\nf01109/Smn\nf01109) displayed elevated numbers of pre-synaptic ghost boutons when compared to wild-type.(0.21 MB TIF)Click here for additional data file.Figure S4\npMAD staining of \nvestigalGAL4\n, \nUAS-Smn-RNAi\n transgenic animals. (A–B) 3rd instar wing discs of vestigalGAL4, pWIZ[UAS-Smn-RNAi]N4 animals are stained with antibodies against SMN (red) (A) and pMAD (green) (B). pMAD staining is reduced in the dorsoventral boundary of the wing disc where SMN expression is decreased (see Figure 10 for wild-type control).(1.37 MB TIF)Click here for additional data file.Figure S5\nNMJ analysis of \nSmn\n enhancers. Modification of the NMJ morphology between muscles 6 and 7 in the A2 segment was assayed in the elavGAL4 pWIZ[UAS-Smn-RNAi]C24 background in trans with all identified modifiers using the pre-synaptic (Horseradish peroxidase (HRP)) and post-synaptic (GluRIIA) markers (see Materials and Methods). In the three cases (f04448, d09801 and d00698) that did not show significant phenotypic alteration, the pWIZ[UAS-Smn-RNAi]N13 allele was also used. In this background, strain f04448 and d09801 enhanced, whereas d00698 showed no interaction (data not shown and Figure 7).(0.74 MB TIF)Click here for additional data file.Figure S6\nNMJ analysis of \nSmn\n suppressors. Modification of the NMJ morphology between muscles 6 and 7 in the A2 segment was assayed in the elavGAL4 pWIZ[UAS-Smn-RNAi]C24 background in trans with all identified modifiers using the pre-synaptic (Horseradish peroxidase (HRP)) and post-synaptic (GluRIIA) markers (see Materials and Methods).(0.78 MB TIF)Click here for additional data file.\n\nREFERENCES:\n1. MonaniUR\n2005\nSpinal muscular atrophy: a deficiency in a ubiquitous protein; a motor neuron-specific disease.\nNeuron\n48\n885\n896\n16364894\n2. FrugierTNicoleSCifuentes-DiazCMelkiJ\n2002\nThe molecular bases of spinal muscular atrophy.\nCurr Opin Genet Dev\n12\n294\n298\n12076672\n3. 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+}

batch_11/PMC2527823.json

@@ -0,0 +1,4 @@
+{
+    "id": "PMC2527823",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2527823\nAUTHORS: K Kato, N K Long, H Makita, M Toida, T Yamashita, D Hatakeyama, A Hara, H Mori, T Shibata\n\nABSTRACT:\nRECK is a novel tumour suppressor gene that negatively regulates matrix metalloproteinases (MMPs) and inhibits tumour invasion, angiogenesis and metastasis. In the present study, we investigated the effects of epigallocatechin-3-gallate (EGCG), a major polyphenol in green tea, on the methylation status of the RECK gene and cancer invasion in oral squamous cell carcinoma cell lines. Our results showed that treatment of oral cancer cells with EGCG partially reversed the hypermethylation status of the RECK gene and significantly enhanced the expression level of RECK mRNA. Inhibition of MMP-2 and MMP-9 levels was also observed in these cells after treatment with EGCG. Interestingly, EGCG significantly suppressed cancer cell-invasive ability by decreasing the number of invasive foci (P<0.0001) as well as invasion depth (P<0.005) in three-dimensional collagen invasion model. Although further investigation is required to assess the extent of contribution of RECK on MMPs to the suppression of invasive behaviour, these results support the conclusion that EGCG plays a key role in suppressing cell invasion through multiple mechanisms, possibly by demethylation effect on MMP inhibitors such as RECK.\n\nBODY:\nCancer development is a multistage process requiring progressive genetic and epigenetic changes in neoplastic and responding stromal cells. During the process of malignant progression, migration of cells into the underlying extracellular matrices is a fundamental feature of tumour invasion. The reversion-inducing cysteine-rich protein with Kazal motifs (RECK), a novel matrix metalloproteinases (MMPs) inhibitor, was originally isolated as a transformation suppressor gene against activated ras oncogenes (Takahashi et al, 1998; Oh et al, 2001; Sasahara et al, 2002; Noda et al, 2003; Simizu et al, 2005; Chang et al, 2006). Previous studies have revealed that RECK is able to inhibit tumour angiogenesis, invasion, and metastasis. Its downregulation has been shown in several types of human cancers. Recently, the decrease in RECK expression is reported to correlate with hypermethylation of the promoter region (Furumoto et al, 2000; Song et al, 2006; Chang et al, 2007; Cho et al, 2007). In human cancers, aberrant methylation of tumour suppressor genes is of comparable significance to classic genetic mutations (Lund and van Lohuizen, 2004; Ha and Califano, 2006; Stresemann et al, 2006; Shaw, 2006). The study of the patterns of gene silencing because of hypermethylation would therefore help to understand and predict cancer cell behaviour and responsiveness to various treatments of cancers (Hellebrekers et al, 2007; Shaw et al, 2007).Tea is the most widely consumed beverage worldwide. Tea components, especially green and black tea constituents, have been reported to prevent carcinogenesis in vitro and in vivo (Jung and Ellis, 2000; Benelli et al, 2002; Yang et al, 2002; Chung et al, 2003; Fang et al, 2003; Fassina et al, 2004; Ju et al, 2005; Khan et al, 2006). Epigallocatechin-3-gallate (EGCG), the major polyphenol in green tea, is believed to be a key active ingredient. Previous studies have shown that EGCG is methylated by catechol-O-methyltransferase and inhibits DNA methyltransferase (DNMT). The inhibition of DNMT would block the hypermethylation of the newly synthesised DNA strand, resulting in the reversal of the hypermethylation and the re-expression of the silenced genes (Fang et al, 2003; Lund and van Lohuizen, 2004; Ha and Califano, 2006; Stresemann et al, 2006; Shaw, 2006; Hellebrekers et al, 2007; Shaw et al, 2007). Other DNMT inhibitors such as 5-aza-2′-deoxycytidine (5-aza-dC) and zebularine also have similar inhibitory effect. Although there is a high potential for developing this group of inhibitors for cancer therapy, side effects and toxicity are serious concerns. The chemoprevention of cancer by tea components as natural inhibitors of DNMT is therefore such a promising approach with less side effects and toxicity (Chung et al, 2003; Lambert and Yang, 2003; Szyf et al, 2004; Ju et al, 2005; Wilson et al, 2007).In the present study, we determined the effects of EGCG treatment on the methylation status and expression level of the RECK gene in human oral squamous cell carcinoma cell lines. The inhibition of oral carcinoma invasion by EGCG was also examined by a three-dimensional collagen invasion model.Materials and methodsCell lines and cell culturesFour human oral squamous cell carcinoma cell lines HSC3, HSC4, SCC9, SCC25 and human cervical cancer cell line HeLa were examined. These cell lines were obtained from Cell Resource Center for Biomedical Research (Tohoku University, Sendai City, Japan). All cell lines were maintained in RPMI-1640 (Sigma-Aldrich Company, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS) (Life Technologies Inc., Gaithersburg, MD, USA) and 50 000 U penicillin, 50 mg streptomycin at 37°C in a 5% CO2 humidified atmosphere. The cancer cell lines were also cultured in medium with 50 μM EGCG (Wako Pure Chemical Industries, Ltd., Osaka, Japan) or 8.7 μM 5-aza-dC (MP Biomedicals, LLC, Eschwege City, Germany) for 6 days and harvested for further analyses as previously described (Fang et al, 2003). To determine the dose-dependent changes, SCC9 and HSC3 cell lines were treated with 5, 10, 20, or 50 μM of EGCG or 8.7 μM of 5-aza-dC for 6 days. EGCG or 5-aza-dC was added, in new culture medium, to the cells on days 1, 3, and 5. For the time course study, the cells were treated with 50 μM of EGCG for 36, 72, or 144 h.Bisulphite modification and methylation-specific PCROne microgram of the purified DNA was subjected to bisulphite modification. Bisulphite modification was performed using CpGenome™ DNA Modification Kit (Chemicon International, Temecula, CA, USA) according to the manufacturer's instructions. For MSP, the following primer sets were used: for methylated DNA, MF_RECK (5′-GTTAGTTTTTTTTTTTATTTTAGTGGTTCGA-3′) and MR_RECK (5′-TCCAAAACCTCCCGAAAACGAAAACG-3′), and for unmethylated DNA, UF_RECK (5′-GGTTAGTTTTTTTTTTTATTTTAGTGGTTTGA-3′) and UR_RECK (5′-ATTTCCAAAACCTCCCAAAAACAAAAACA-3′). Reactions were performed in 20 μl volumes under the following conditions: 95°C for 10 min; then 40 cycles of 95°C for 30 s, 56°C for 30 s, and 72°C for 30 s; and finally 7 min at 72°C. The PCR product lengths for methylated and unmethylated RECK are 201 and 205 bp. CpGenome Universal Methylated DNA (Serologicals, Atlanta, Georgia, USA) and normal human blood DNA was used as positive control for methylated and unmethylated status. Water blank was used as a negative control. Positive and negative controls worked appropriately in each round of PCR. All assays were performed in triplicate.Reverse transcription–PCR and quantitative real-time PCRTotal RNA was isolated from 105 to 106 cultured cells using a Trizol reagent kit (Invitrogen, Carlsbad, CA, USA). cDNA was synthesised from 1 μg of total RNA using MMLV Reverse Transcriptase (Invitrogen) with random hexamers. RECK cDNA was amplified by PCR using the sense primer 5′-GCAGGGGAAGTTGGTTGTTA-3′ and antisense primer 5′-TGCCAGCAAAACAAGAACAG-3′. Reactions were performed in 20 μl volumes under the following conditions: 95°C for 10 min; then 35 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s; and finally 7 min at 72°C. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control to estimate the efficiency of the cDNA synthesis in each cell line with forward primer: 5′-AGCATCTACACCTGAGGACAAGAC-3′; reverse primer: 5′-TTTTGCTCTTAACCACGTTTATTGA-3′. The integrated optical density of each band was quantified by densitometry. The relative levels of RECK mRNA were normalised against the GAPDH.Quantitative real-time PCR assay was performed using SYBR®Premix Ex Taq™ in Thermal Cycler Dice® (Takara, Tokyo, Japan). The cycling conditions were initial denaturation at 95°C for 10 s, and 40 cycles at 95°C for 5 s, 60°C for 30 s according to the SYBR®Premix Ex Taq (Perfect Real Time) protocol. Second derivative maximum method was used to calculate the Ct (threshold cycle) value and standard curve method was used for relative quantification analysis. The Ct value of RECK was normalised by the Ct of GAPDH in the same sample. Each reaction was run in triplicate.Gelatin zymographyCancer cell lines were seeded in bio-coat culture disks (Becton Dickinson Labware, Bedford, MA, USA) for 6 days. Fresh medium without FBS was replaced 24 h before analysis. MMP-2 and MMP-9 enzymatic activity in cancer cell lines was determined by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE) gelatin zymography as described in our previous study (Kato et al, 2005). In brief, the gelatinolytic activity was measured in the presence of both proforms and activated forms of the MMP-2 and MMP-9. The cell extract was diluted in the ratio of 1 : 1 with sample buffer (TEFCO, Tokyo, Japan) and left at room temperature for 10 min. The cell extracts were applied to SDS–PAGE using a 10% gel containing 0.1% gelatin. After electrophoresis the gel was rinsed with renaturing buffer (TEFCO) for 30 min, and the gel was incubated in developing buffer (TEFCO) for 48 h at 37°C. After incubation the gel was stained with 0.05% Coomassie brilliant blue R250 (Wako Pure Chemical Industries, Osaka, Japan). The MMPs were detected as transparent bands on the blue background of the Coomassie blue stained slab gel.Collagen gel invasion modelCollagen gels were used as matrices for cancer cell invasion and prepared according to the method of Hase et al (2006) with a little adjustment. Type I collagen (Nippon Meat Packers Inc., Osaka, Japan) was mixed with medium and × 10 PBS at a ratio of 1 : 1 : 8 and air-vacuumed for 30 min before incubated in 5% CO2 and 37°C until gelling was completed. Cancer cells (3 × 105) from each cell line were seeded on the collagen gels in medium with and without EGCG or 5-aza-dC. The cancer cells were maintained at 5% CO2 atmosphere and 37°C for 7 and 14 days. The collagen gel was collected and fixed in 10% formalin, embedded in paraffin, stained with haematoxylin and eosin and examined for cancer cell invasion.Assays for cell invasion and migrationCell invasion and immigration assays were used to assess the formation of invasive foci and the depth of cell invasion into the collagen matrix previously described by Hotary et al (2000) and Liebersbach and Sanderson (1994). Six days after the addition of cells to gels, invasive foci were counted in randomly selected fields at × 200 magnification on phase-contrast microscope. The depth of cell invasion was determined by measuring the distance from the top of the gel to the leading front of migrating cells. The leading front distance was defined as the point at which two of the most distantly migrating cancer cells were simultaneously in focus in one field under × 200 magnification. Measurements were made using the calibrated micrometer of a Nikon inverted microscope. Measurements were taken in five fields within each well, and the mean distance was determined.To measure cell proliferation rates, 8 × 104 cells were placed into each culture plate and cultured for 3, 5, 7 days in medium without and with 50 μM EGCG and 8.7 μM 5-aza-dC. At each time point, the cells were harvested by trypsinisation and counted using a hemacytometer.Cell migration ability was assessed by seeding 2–5 × 105 cells atop collagen gels with surfaces partially covered by glass coverslips (1 × 2 cm; Matsunami Glass Ind, Ltd., Osaka, Japan). When the cells reach confluence, the glass fragments were removed, leaving a cell-free area on the gel. At this time, medium was added to all cultures in the absence or presence of EGCG and 5-aza-dC. The distances migrated across the gels were observed 24 h later using an inverted microscope.Statistical analysisDifferences between treatment and control groups were assessed by analysis of variance with post hoc test (Dunnett's test). Statistical analyses on the invasive foci and depth of invasion of the cancer cell lines were performed using the Student's t-test. The results were considered statistically significant at P<0.05.ResultsMethylation status and expression of the RECK gene in OSCC cell linesA hypermethylated RECK promoter was observed in all four OSCC cell lines (100%) by MSP. SCC9 and SCC25 cell lines contained both unmethylated and methylated promoters whereas HSC4 and HSC3 cell lines had strongly methylated promoter and faintly unmethylated promotor. Methylation of RECK gene was not detected in HeLa cancer cell line whereas its unmethylation-specific band appeared as a weak band (Figure 1A). The expression levels of RECK mRNA in 4 human oral cancer cell lines and HeLa were examined by RT–PCR. The results showed that the HSC3 and HSC4 cell lines expressed very low RECK mRNA levels. SCC9 and SCC25 cells had downregulated expression whereas HeLa produced a normal amount of RECK mRNA (Figure 1A).Reversal of hypermethylation status and enhanced expression of RECK gene in OSCC cell lines by EGCGTo evaluate whether methylation status of RECK promoter is associated with transcriptional downregulation of RECK gene in the OSCC cell lines, demethylation treatment by 5-aza-dC and EGCG were performed on the OSCC cell lines. In all cancer cell lines, the RECK gene had hypermethylation status with the low level of the respective mRNA expression. The appearance of unmethylation-specific bands of RECK gene in all four cancer cell lines became more intense after treatment with 50 μM EGCG or 8.7 μM 5-aza-dC for 6 days. Treatment of SCC9 and HSC3 with EGCG and 5-aza-dC for 6 days also enhanced the transcription of RECK mRNA whereas RECK mRNA level was not significantly altered in HSC4 and SCC25 after treatment with EGCG (Figure 1B).We examined the time-dependent and dose-dependent effects of EGCG in SCC9 and HSC3 cell lines. After treating cells with 5, 10, 20, or 50 μM of EGCG for 6 days, the methylation-specific bands of this gene still existed but in a weak appearance. The unmethylation-specific bands of RECK gene, however, appeared to be enhanced after treatment with 20 or 50 μM of EGCG for 6 days. Corresponding to the appearance of the unmethylation-specific band was the increase of expression of RECK mRNA by conventional RT–PCR. The unmethylation-specific bands and mRNA expression were significantly stronger with 50 μM of EGCG in comparison with those treated with 20 μM EGCG or lower concentrations (P<0.01). The reversal of hypermethylation and increase of expression of RECK by EGCG were almost similar to that produced by the classical DNMT inhibitor, 5-aza-dC. After treating the cells with 50 μM of EGCG for 36, 72, and 144 h, strong unmethylation-specific bands of the RECK gene began to appear at 72 h. The higher level of mRNA expression of RECK was also observed at 72 and 144 h (P<0.01) (Figure 2).To confirm the effect of EGCG on RECK expression, real-time quantitative PCR was performed to determine the mRNA expression level of RECK gene in HSC3 and SCC9 cell lines after treatment with different concentrations of EGCG. The results showed that the relative amount of mRNA expression of RECK was increased in a dose- and time-dependent manner with significant effect at 20 and 50 μM EGCG (P<0.01 and P<0.001) and at 72 and 144 h (P<0.01 and P<0.001). This result was generally consistent with those from general reverse transcription–PCR, however, with a significant effect at lower concentration of EGCG (20 μM) (Figure 3).Downregulation of MMPs by EGCGGelatin zymography results showed that MMP-2 and MMP-9 expression and activity were suppressed in SCC9 and HSC3 cells treated with EGCG and 5-aza-dC for 6 days (Figure 4). Culture with EGCG or 5-aza-dC decreased total MMP-2 levels and downregulated the activation of proMMP-2. Although proMMP-9 was detectable, active forms of MMP-9 were not readily observed in this experiment. Treatment with 50 μM EGCG did not cause downregulation in MMP-9 activity but MMP-2 in HSC4 and SCC25 cancer cell line (data not shown).Inhibition of cancer invasion in collagen matrices by EGCGWhen cultured atop collagen gels, SCC9, SCC25, and HeLa cells formed confluent monolayers that remained confined to the surface of the underlying gel for the entire culture period regardless of the presence of EGCG or 5-aza-dC. We also did not observe the significant invasion in the SCC9 and SCC25 cells. However, treatment with EGCG significantly inhibits cancer-invasive ability in collagen model in HSC4 and HSC3 cancer cell lines (Figure 5A–D). During 7 days treatment with EGCG, HSC3 and HSC4 cells proliferated at a lower rate compared with those of control or 5-aza-dC treated cells. Although treatment with 50 μM EGCG caused some damaged cells with vesicles, signs of toxicity were not apparent at lower doses.Our results indicated that treatment with 50 μM EGCG significantly inhibited cancer invasion in a three-dimensional collagen model. Furthermore, EGCG or 5-aza-dC significantly blocked cancer invasion in HSC3 cells by decreasing the mean number of invasive foci/field (P<0.0001 and P<0.00001, respectively). Results are shown as the mean number of invasive foci ±1 s.d. in five randomly selected fields in HSC3 cells treated with 5-aza-dC, EGCG and control (6±2 foci, 11.4±2.7 foci, and 26.4±3.8 foci, respectively). Interestingly, regarding cell migration and depth of invasion, EGCG significantly inhibited cells to invade deeply into the collagen gel compared with control (93±32.3 vs 191.2±33.7 μm, P<0.005) as well as suppressed the cells migrating across the gels surface (Figure 5B, D and 6A, B). Similar effects were also observed in HSC4 cell line (Table 1).In the presence of EGCG, HSC3 and HSC4 cells cultured atop collagen gels for 6 days displayed widespread but shallow foci of invasion. In contrast, control cells (EGCG-free medium) invaded and generated large and deep pits extending well in the collagen matrix. After 14 days, HSC3 cells cultured in the absence of EGCG formed an aggressive invasion and proliferation (with stratified 5–6 cell layers) whereas invasion of HSC3 cells treated with EGCG was markedly inhibited and only generated pits underlying collagen gels (Figure 5C and D).DiscussionIn the present study, we report that EGCG, a major component of green tea, may enhance RECK expression by reversal of hypermethylation of RECK promoter and inhibit MMP activities as well as cancer cell invasion in OSCC cell lines. Recent studies have also shown that RECK methylation is associated with increase of metastasis and invasion in human cancers (Chang et al, 2006, 2007; Cho et al, 2007).Our findings implied that the hypermethylation of RECK is associated with a low level of mRNA expression in oral squamous cell carcinoma cell lines. Of these, SCC9 and SCC25 cell lines were partially methylated whereas HSC4 or HSC3 cells were almost completely methylated. The results showed that treatment of oral cancer cells with EGCG partially reversed the hypermethylation status of the RECK gene and significantly enhanced the expression level of RECK mRNA. EGCG has been reported to reverse hypermethylation and reactivate several tumour suppressor genes in human oesophageal squamous cell carcinoma cell lines at doses from 20 μM (Fang et al, 2003) by nested two-stage MSP. However, present oral cancer cell lines appear to be less susceptible to a demethylation effect by EGCG. Additional work is needed to determine whether different genes respond similarly or differently to the EGCG treatment in different cell lines under various treatment conditions.EGCG has been shown to affect MMP activity both directly and indirectly, in particular MMP-2 at relatively low doses (10–20 μM). EGCG has been reported to inhibit activating protein-1 (AP-1) that regulates MMP expression. In another way, EGCG could also inhibit the proMMP-2 protein secretion by perturbing the intracellular vesicular trafficking (Oh et al, 2004; Khan et al, 2006). However, our data demonstrate that OSCC cell lines-derived MMP-2 and MMP-9 were inhibited at 50 μM of EGCG treatment. Previous studies showed that restored expression of RECK in malignant cells resulted in suppression of invasive activity with concomitant decrease in the secretion of MMPs (Takahashi et al, 1998; Okabe et al, 1999; Liu et al, 2003; Noda et al, 2003). Treatment with EGCG, HSC3 and HSC4 cells cultured on top of collagen gels produced widespread but shallow foci of invasion whereas control cells (EGCG-free medium) invaded and generated large and deep pits extending well in the collagen matrix. This could be because of the ability to regulate MMPs by RECK restored expression. Earlier study has shown that stimulated RECK expression significantly caused downregulation of MMP-2 and MMP-9 activities (Takahashi et al, 1998; Liu et al, 2003; Oh et al, 2004). In experimental systems, cellular invasion is reduced by the presence of endogenous tissue inhibitors of MMPs (TIMPs) and RECK. Recently, TIMP-2 has been reported to enhance expression of RECK through Rap1 signalling resulting in an indirect, time-dependent inhibition of cell migration (Oh et al, 2004). Our findings suggest that EGCG and 5-aza-dC may act simultaneously through different target proteins such as TIMPs and RECK to suppress invasion. Further study is needed to define the extent of contribution of RECK on MMPs to the suppression of cell-invasive behaviours.The results support the conclusion that EGCG plays a crucial role in inhibiting cell invasion of a collagen model. EGCG significantly suppressed cancer-invasive ability in oral cancer cell lines by decreasing the number of invasive foci and depth of invasion as strong as classical DNMT inhibitor, 5-aza-dC. Oral administration of green tea has been reported to inhibit tumour genesis in different organs and multiple mechanisms may be involved (Jung and Ellis, 2000; Khan et al, 2006; Chiang et al, 2006; Okabe et al, 1999). Importantly, these effects of EGCG have been shown to be selective for cancer cells, as normal cells were not affected by this treatment (Khan et al, 2006).The present study indicated that the effective dose of EGCG in inhibiting cancer invasion and migration is at 50 μM. At a concentration of 20 μM or lower, EGCG did not significantly affect cancer invasion or migration of the cells. It is achievable in the oral cavity (in saliva) after drinking green tea and perhaps in the stomach, esophagus and the intestines where there is direct contact between EGCG and the epithelial cells (Fang et al, 2003). This effective concentration is variable in different organs and the DNMT inhibition would depend on the systemic bioactivities and the bioavailability of EGCG in a particular organ site. Inhibition of DMNT is expected to prevent hypermethylation; however, severe inhibition of DNMT activity may cause DNA hypomethylation, genomic instability and other disorders (Szyf et al, 2004; Wilson et al, 2007). The effects of EGCG in vitro have been obtained with relatively high concentrations than observed in the plasma or tissues of animals or in human plasma after the administration of green tea or EGCG (Masuda et al, 2001; Khan et al, 2006). Therefore, it is not clear whether the activities observed with high EGCG concentrations in cell lines can be observed in vivo. More comprehensive studies in animal models and humans are needed to determine the optimal doses and side effects of EGCG.In conclusion, our findings raise the possibility that EGCG could inhibits cancer cell invasion through reversal of hypermethylation status of RECK and downregulation of MMP-2 and MMP-9. Our data together with earlier studies indicate that EGCG as a natural demethylating agent could be a promising therapeutic strategy for the development of combination chemopreventive/chemotherapeutic approaches in oral cancer treatment. Additional investigations are required to fully elucidate the molecular mechanisms by which green tea constituents, and EGCG in particular, inhibit tumour invasion and metastasis.\n\nREFERENCES:\n1. 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Ju J, Hong J, Zhou JN, Pan Z, Bose M, Liao J, Yang GY, Liu YY, Hou Z, Lin Y, Ma J, Shih WJ, Carothers AM, Yang CS (2005) Inhibition of intestinal tumorigenesis in Apcmin/+ mice by (−)-epigallocatechin-3-gallate, the major catechin in green tea. Cancer Res\n65: 10623–1063116288056\n15. Jung YD, Ellis LM (2000) Inhibition of tumour invasion and angiogenesis by epigallocatechin gallate (EGCG), a major component of green tea. Int J Exp Path\n82: 309–316\n16. Kato K, Hara A, Kuno T, Kitaori N, Huilan Z, Mori H, Toida M, Shibata T (2005) Matrix metalloproteinases 2 and 9 in oral squamous cell carcinomas: manifestation and localization of their activity. J Cancer Res Clin Oncol\n131: 340–34615614523\n17. Khan N, Afaq F, Saleem M, Ahmad N, Mukhtar H (2006) Targeting multiple signaling pathways by green tea polyphenol (−)-epigallocatechin-3-gallate. Cancer Res\n66: 2500–250516510563\n18. Lambert JD, Yang CS (2003) Mechanisms of cancer prevention by tea constituents. J Nutr\n133: 3262–3267\n19. Liebersbach BF, Sanderson RD (1994) Expression of Syndecan-1 inhibits cell invasion in to type I collagen. J Biol Chem\n269: 20013–200198051085\n20. Liu LT, Chang HC, Chiang LC, Hung WC (2003) Histone deacetylase inhibitor up-regulates RECK to inhibit MMP-2 activation and cancer cell invasion. Cancer Res\n63: 3069–307212810630\n21. Lund AH, van Lohuizen M (2004) Epigenetics and cancer. Genes Dev\n18: 2315–233515466484\n22. Masuda M, Suzui M, Weinstein IB (2001) Effects of epigallocatechin-3-gallate on growth, epidermal growth factor receptor signaling pathways, gene expression, and chemosensitivity in human head and neck squamous cell carcinoma cell lines. Clin Cancer Res\n7: 4220–422911751523\n23. Noda M, Oh J, Takahashi R, Kondo S, Kitayama H, Takahashi C (2003) RECK: a novel suppressor of malignancy linking oncogenic signaling to extracellular matrix remodeling. Cancer Metastasis Rev\n22: 167–17512784995\n24. Oh J, Seo DW, Diaz T, Wei B, Ward Y, Ray JM, Morioka Y, Shi S, Kitayama H, Takahashi C, Noda M, Stetler-Stevenson WG (2004) Tissue inhibitors of metalloproteinase 2 inhibits endothelial cell migration through increased expression of RECK. Cancer Res\n64: 9062–906915604273\n25. Oh J, Takahashi R, Kondo S, Mizoguchi A, Adachi E, Sasahara RM, Nishimura S, Imamura Y, Kitayama H, Alexander DB, Ide C, Horan TP, Arakawa T, Yoshida H, Nishikawa S, Itoh Y, Seiki M, Itohara S, Takahashi C, Noda M (2001) The membrane-anchored MMP inhibitor RECK is a key regulator of extracellular matrix integrity and angiogenesis. Cell\n107: 789–80011747814\n26. Okabe S, Ochiai Y, Aida M, Park K, Kim SJ, Nomura T, Suganuma M, Fujiki H (1999) Mechanistic aspects of green tea as a cancer preventive: Effect of components on human stomach cancer cell lines. Jpn J Cancer Res\n90: 733–73910470285\n27. Sasahara RM, Brochado SM, Takahashi C, Oh J, Maria-Engler SS, Granjeiro JM, Noda M, Sogayar MC (2002) Transcriptional control of the RECK metastasis/angiogenesis suppressor gene. Cancer Detection Prev\n26: 435–443\n28. Shaw R (2006) The epigenetics of oral cancer. Int J Oral Maxillofac Surg\n35: 101–10816154320\n29. Shaw RJ, Hall GL, Lowe D, Bowers NL, Liloglou T, Field JK, Woolgar JA, Risk JM (2007) CpG island methylation phenotype (CIMP) in oral cancer: Associated with a marked inflammatory response and less aggressive tumour biology. Oral Oncol\n43: 878–88617257884\n30. Simizu S, Takagi S, Tamura Y, Osada H (2005) RECK-mediated suppression of tumor cell invasion is regulated by glycosylation in human tumor cell lines. Cancer Res\n65: 7455–746116103099\n31. Song SY, Son HJ, Nam E, Rhee JC, Park C (2006) Expression of reservesion-inducing-cystein-rich protein with Kazal motifs (RECK) as prognostic indicator in gastric cancer. Eur J Cancer\n42: 101–10816324834\n32. Stresemann C, Brueckner B, Musch T, Stopper H, Lyko F (2006) Functional diversity of DNA methyltransferase inhibitors in human cancer cell lines. Cancer Res\n66: 2794–280016510601\n33. Szyf M, Pakneshan P, Rabbani SA (2004) DNA demethylation and cancer: therapeutic implications. Cancer Lett\n211: 133–14315219937\n34. Takahashi C, Sheng Z, Horan TP, Kitayama H, Maki M, Hitomi K, Kitaura Y, Takai S, Sasahara RM, Horimoto A, Ikawa Y, Ratzkin BJ, Arakawa T, Noda M (1998) Regulation of matrix metalloproteinase-9 and inhibition of tumor invasion by the membrane-anchored glycoprotein RECK. Proc Natl Acad Sci USA\n95: 13221–132269789069\n35. Wilson AS, Power BE, Molly PL (2007) DNA hypomethylation and human diseases. Biochim Biophys Acta\n1775: 138–16217045745\n36. Yang CS, Maliakal P, Meng X (2002) Inhibition of carcinogenesis by tea. Annu Rev Pharmacol Toxicol\n42: 25–5411807163"
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+    "id": "PMC2527827",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2527827\nAUTHORS: H Y Sheikh, J W Valle, T Waddell, K Palmer, G Wilson, A Sjursen, O Craven, R Swindell, M P Saunders\n\nABSTRACT:\nTegafur–uracil (UFT) plus leucovorin® (LV, folinic acid) with alternating irinotecan and oxaliplatin were effective and well tolerated in patients with metastatic colorectal cancer (mCRC) in a phase I study. This study expanded the maximum tolerated dose group. Patients aged ⩾18 years had histologically confirmed, inoperable, previously untreated, measurable mCRC. Patients received irinotecan 180 mg m−2 on day 1, oxaliplatin 100 mg m−2 on day 15 and UFT 250 mg m−2 plus LV 90 mg on days 1–21 every 28 days. The phase I/II study comprised 45 patients, 29 at the maximum tolerated dose (MTD). The response rate in 38 evaluable patients was 63% (95% confidence interval (CI): 49–80). Median time to progression and overall survival were 8.7 months (95% CI: 7.9–10.4) and 16.8 months (95% CI: 9.6–25.3), respectively. In the MTD group, one patient had grade 3 leucopaenia; one had grade 3 neutropaenia; three had grade 3 diarrhoea; and one had grade 3 neurotoxicity. No hand–foot syndrome grade >1 was seen. In total, 67% of eligible patients received second-line therapy. UFT plus LV with alternating irinotecan and oxaliplatin is an efficacious first-line treatment for mCRC, with minimal neurotoxicity and hand–foot syndrome.\n\nBODY:\nPatients with unresectable metastatic colorectal cancer (mCRC) have a poor prognosis in the absence of effective chemotherapy. The foundation of treatment for these patients is 5-fluorouracil (5-FU) with leucovorin® (LV), either alone or in combination with oxaliplatin (FOLFOX) or irinotecan (FOLFIRI). Many patients with mCRC receive only first-line treatment (Table 1), with some refusing second-line therapy and preferring to preserve their quality of life, whereas others are not offered this option as they are considered unfit as a result of disease progression.Optimising first-line treatment is essential in mCRC. As exposure to three active agents, rather than second-line therapy itself, appears to predict improved survival (Grothey and Sargent, 2005), ‘up-front’ administration of three effective drugs may be the most effective way to improve outcomes. Consequently, several groups have investigated the FOLFOXIRI combination (5-FU, oxaliplatin and irinotecan) in patients with mCRC (Masi et al, 2004; Souglakos et al, 2006; Falcone et al, 2007). Results from phase III studies have been conflicting, however, with Falcone et al (2007) demonstrating better outcomes for patients treated with FOLFOXIRI compared with FOLFIRI, and Souglakos et al (2006) reporting no significant difference between the two regimens. Nonetheless, extremely promising overall survival (OS) rates were reported in these studies (22.6 months and 21.5 months, respectively), and a triple-drug approach holds great promise, provided the triplet regimen is tolerable.Toxicity is a significant problem with 5-FU-based regimens. Intravenous (i.v.) bolus 5-FU is associated with considerable myelosuppression (Meta-Analysis Group In Cancer, 1998). Continuous infusion 5-FU, although less toxic, requires venous access using a tunnelled central line and portable infusion pumps, which are inconvenient for patients, while indwelling catheters can cause infection and thrombosis (Puig-la Calle et al, 1996; Verso and Agnelli, 2003). The oral fluoropyrimidine tegafur–uracil (UFT) is a convenient and well tolerated alternative to i.v. 5-FU. In phase III studies, UFT with LV had equivalent efficacy compared to that of i.v. 5-FU/LV, but with significantly better tolerability (Carmichael et al, 2002; Douillard et al, 2002). In both studies, UFT with LV was associated with significantly fewer haematological adverse events, including febrile neutropaenia, than bolus 5-FU/LV. In addition, patients in the UFT with LV group had a significantly lower incidence of stomatitis and other gastrointestinal events than 5-FU/LV. A number of small studies have shown that UFT can be combined with oxaliplatin (Feliu et al, 2004; Rosati et al, 2005; Bennouna et al, 2006; Bajetta et al, 2007a) or irinotecan (Mendez et al, 2005; Delord et al, 2007; Bajetta et al, 2007a), both combinations being effective and well tolerated in first-line mCRC.The present phase I/II study, SCOUT (study of CPT-11, oxaliplatin, UFT triple therapy), evaluated the efficacy and tolerability of UFT with LV plus alternating irinotecan and oxaliplatin in chemonaive patients with mCRC. We hypothesised that alternating oxaliplatin and irinotecan would allow patients to benefit from concurrent treatment with all three drugs as soon as they were diagnosed with metastatic disease, while allowing them to recover from adverse events associated with each drug before it was administered again. Results from the phase I study have been published in full (Sheikh et al, 2007). Here we present results for the expanded group of patients treated at the maximum tolerated dose (MTD) and overall results from the phase I/II study.Materials and methodsStudy design and patientsThis single-centre, open-label, non-randomised phase I/II study was conducted at the Christie Hospital (Manchester, UK) on patients with histologically confirmed, metastatic adenocarcinoma of the colon or rectum, with inoperable, measurable metastatic disease. Inclusion and exclusion criteria have been described previously (Sheikh et al, 2007). In brief, patients were aged ⩾18 years, had no prior chemotherapy for metastatic disease and a World Health Organization performance status of 0–2. All patients had adequate organ and haematological function.The trial was conducted with full approval of the local ethical committee, according to accepted standards of good clinical practice, and in agreement with the latest version of the Declaration of Helsinki. All patients provided written informed consent.TreatmentIn the phase I study, patients received irinotecan 180 mg m−2 as a 90 min infusion on day 1 of the 28-day cycle, oxaliplatin 85–100 mg m−2 as a 2 h infusion on day 15 and UFT capsules 200–300 mg m−2 d−1 taken orally with LV 90 mg d−1 in three divided doses on days 1–21. The MTD was irinotecan 180 mg m−2, oxaliplatin 100 mg m−2 and UFT 250 mg m−2 plus LV 90 mg. Prophylactic antiemetics (dexamethasone 8 mg and ondansetron 8 mg) were administered intravenously with the irinotecan and oxaliplatin infusions and thereafter orally for 48 h.Chemotherapy was administered for at least 8 weeks (two cycles) before radiological reassessment, unless a criterion for study discontinuation was met. Patients remained on treatment until clinical or radiological progression, the occurrence of unacceptable or cumulative toxicity or withdrawal was requested by the patient or investigator. Treatment was continued for a further 2 months in patients with signs of clinical benefit (stable disease or complete or partial response) up to 6 months. Selected patients could continue treatment for longer than 6 months at the investigator's discretion, and could receive further treatment with the SCOUT regimen or another treatment of the investigator's choice. Re-treatment with SCOUT was only allowed in patients who had not progressed during the initial treatment period.Dose reductions and delaysChemotherapy was delayed by 1 week in patients with neutropaenia grade ⩾2 or platelets <100 × 109 l−1 (<75 × 109 l−1 for oxaliplatin). If there was more than one delay or if a delay lasted ⩾2 weeks, doses of irinotecan and oxaliplatin were reduced by 20% and the daily UFT dose was reduced by one capsule (100 mg). Treatment continued at the lower dose for subsequent cycles unless further toxicity occurred. If a further delay for myelotoxicity occurred, 50% reduction was made to the original irinotecan and oxaliplatin doses. Withdrawal of the patient from the study was considered if their performance status had deteriorated.Delayed diarrhoea was treated early and aggressively with loperamide, with the addition of oral ciprofloxacin if it persisted for >24 h. However, after grade 3/4 diarrhoea, treatment was delayed until complete recovery, and then resumed at 80% of the irinotecan dose and with the UFT daily dose reduced by one capsule. If diarrhoea of any grade had not resolved by the next cycle, treatment was delayed by 1 week. If further grade ⩾3 diarrhoea occurred, irinotecan was reduced to 50% of the original dose and the daily UFT dose was reduced by two capsules. Oxaliplatin was omitted for grade ⩾3 paraesthesia of hands or feet and dysaesthesia in the throat. Any significant deterioration in liver or renal function was investigated by ultrasound examination to rule out reversible biliary or renal outflow obstruction.Response and toxicity evaluationPatients were assessed clinically every 14 days and a full blood count and biochemistry profile performed. Serum tumour carcinoembryonic antigen (CEA) measurements were performed monthly if raised at baseline. Toxicities were recorded at 2-weekly visits according to the National Cancer Institute Common Toxicity Criteria (version 2) and the dose-limiting toxicities at 1 month, that is, diarrhoea, lethargy and vomiting, used to decide the dose escalation schedule during the phase I study, as reported previously (Sheikh et al, 2007).Radiological assessment by CT scan was performed after two cycles using the Response Evaluation Criteria in Solid Tumors (RECIST) (Therasse et al, 2000). All scans were performed by a dedicated gastrointestinal radiologist who provided measurements of marker lesions. From these measurements, response was confirmed by two research fellows. In cases of disagreement, scans were reviewed by the principal investigator. Only patients receiving two full 28-day cycles were assessable for the primary endpoint, that is, the objective response rate (ORR), defined as the number of patients achieving a partial or complete response at 8 weeks.ResultsPatient characteristicsOverall, 45 patients were treated, 29 of whom received the MTD. Patient demographics and clinical characteristics are shown in Table 2. In total, seven patients did not receive two cycles of SCOUT and were not evaluable for response assessment; four of these patients were in the MTD group. The evaluable population therefore comprised 38 patients in total and 25 patients treated at the MTD. Overall, five patients were not assessable for time to progression (TTP), two of whom were treated at the MTD (three patients died before completing an assessment scan after two cycles and two withdrew for psycho-social reasons). All patients were assessable for toxicity and OS. A majority of patients had multiple lesions; only six patients had disease confined to the liver.Response to treatmentResponse to treatment is shown in Table 3.An ORR of 68% (95% CI: 46–85%) and a disease-control rate of 100% (95% CI: 86–100%) were observed in the 25 evaluable patients who received the MTD. Median OS was 19.6 months in 29 assessable patients (95% CI: 15.5–27.2) (Figure 1A) and median TTP was 8.5 months in 27 assessable patients (95% CI: 7.6–11.1) (Figure 1B). One-year survival was 72.4% (95% CI: 52.3–85.1%) and 2-year survival was 37.2% (95% CI: 13.2–61.8%).The ORR in the 38 evaluable patients in the phase I/II study was 63% (95% CI: 46–78%), with a disease-control rate of 89% (95% CI: 75–97%). After a median follow-up of 14.9 months, median OS was 16.8 months (95% CI: 9.6–25.3) in 45 evaluable patients (Figure 1C) and median TTP was 8.7 months (95% CI: 7.9–10.4) in 40 evaluable patients (Figure 1D). One-year survival was 62.2% (95% CI: 46.4–74.6%) and 2-year survival was 38.7% (95% CI: 21.6–55.5%).Liver resections were performed on three patients. One patient with a partial response after four cycles underwent an R0 resection, one patient who had stable disease after three cycles had an R1 resection and one patient who had 5 cycles and achieved a partial response had an R1 resection. Another patient, who underwent a total of 11 treatment cycles and achieved a partial response on a re-challenge with SCOUT is being assessed for liver resection.TolerabilityTolerability data for the phase I cohort are detailed in our earlier publication. In the MTD group, 138 UFT courses were prescribed to the 29 patients, 16 of which were reduced by 1 capsule daily and 8 of which were reduced by 2 capsules daily; 21 patients (72%) received UFT without a dose reduction, 13 of whom (45%) received all 6 UFT cycles at full dose. A total of 139 doses of irinotecan therapy were administered, 17 doses were reduced by 20% and 3 by 50%; 132 doses of oxaliplatin were administered, 9 were reduced by 20% and 5 by 50%. The median dose intensity was 89% for both irinotecan and oxaliplatin after two cycles; 92% and 91% for irinotecan and oxaliplatin, respectively, after four cycles; and 95% and 92% for irinotecan and oxaliplatin, respectively, after six cycles.SCOUT was well tolerated at the MTD, as shown in Table 4. One patient developed grade 3 neutropaenia and another had grade 3 leucopaenia. One patient with a history of hypertension, atrial fibrillation and type II diabetes had a grade 4 cardiac event and died following a myocardial infarction before cycle four. Alopecia and neurotoxicity were minimal: three patients (10%) had grade 2 alopecia and three (10%) had grade 2/3 neurotoxicity. Hand–foot syndrome grade >1 was not observed.Second-line therapyAfter failure of first-line therapy, 24 out of the 29 patients (83%) treated at the MTD were considered for second-line treatment; 3 patients had not progressed at the time of the analysis and a further 2 patients were excluded as they were not assessable for progression, as described above. Six patients (25%) were deemed unfit to receive second-line therapy. Eighteen patients received second-line therapy (Table 5). Nine patients (38%) resumed SCOUT, eight of whom were assessable for response: one patient had a complete response (13%); three had partial responses (38%); one had stable disease (13%) and three had progressive disease (38%). Therefore, the ORR to re-treatment with SCOUT in the MTD group was 50% and the disease control rate was 63%. Twelve patients received other second-line regimens, including capecitabine-, irinotecan- and oxaliplatin-based regimens, as detailed in Table 5.If patients treated at all doses are considered, 36 out of the 45 patients (80%) were considered for second-line therapy, 12 (33%) of whom were deemed unfit for further chemotherapy. Twelve (50%) of the 24 patients who received second-line chemotherapy resumed SCOUT. Two of the additional three patients from the phase I study who resumed SCOUT responded to the re-challenge with stable disease, and one had progressive disease. Therefore, the ORR to re-treatment with SCOUT chemotherapy in all patients was 36% and the disease control rate was 64%.Nine out of the 12 patients (75%) who received second-line SCOUT went on to receive a third-line chemotherapy regimen, as shown in Table 5.Out of the remaining 12 patients who received second-line therapies other than SCOUT, 3 (25%) went on to receive a third-line agent (mitomycin C/capecitabine: n=2; XELOX: n=1).DiscussionRecent studies have demonstrated that triple-drug regimens can improve survival and response in patients with mCRC. Many of these regimens, however, can be associated with excessive toxicities. Alternating therapy, in which only two out of the three drugs are administered at one time, provides a means of delivering three effective drugs while minimising toxicity. The results from the present study confirm that alternating irinotecan and oxaliplatin during treatment with UFT with LV allows patients to receive an effective triple-drug regimen without the excessive haematological toxicities often observed with such treatments.The combination of UFT with LV plus irinotecan and oxaliplatin was highly effective, with excellent ORRs that were consistent with the phase I study results and better than those reported for UFT with LV plus irinotecan (Mendez et al, 2005; Delord et al, 2007; Bajetta et al, 2007a) or UFT with LV plus oxaliplatin (Feliu et al, 2004; Rosati et al, 2005; Bennouna et al, 2006; Bajetta et al, 2007a), providing further support for the up-front treatment approach. Particularly notable was the fact that all patients treated at the MTD benefited from therapy, with partial responses in 68% patients and stable disease in 32%. The OS of 19.6 months seen in patients treated at the MTD was comparable with results obtained for other triplet regimens (Table 1).Alternating oxaliplatin and irinotecan every 2 weeks allowed relatively high doses of both agents to be used without significant toxicities. Alopecia and neurotoxicity, which are commonly observed in patients treated with irinotecan and oxaliplatin, were minimal in our patients. Grade 2 alopecia was observed in three patients (7%) and only one patient had grade 3 neuropathy. Another feature of this study was the very low incidence of grade 3/4 haematological toxicities. This is likely to be a result of the alternating schedule, in which a 4-week gap between each dose of irinotecan and each dose of oxaliplatin allowed patients to recover from oxaliplatin- and irinotecan-induced toxicities before each drug was administered again. Indeed, at the MTD, only one patient had grade 3 neuropathy. Grade 3/4 toxicities were mainly gastrointestinal, with grade 3 diarrhoea in 10% patients. The single grade 4 adverse event observed at the MTD was an unrelated cardiac event in a patient with a history of hypertension, atrial fibrillation and type II diabetes. Hand–foot syndrome, a disturbing and disabling condition that can impact the quality of life of affected patients (Scheithauer and Blum, 2004), is often seen in patients treated with 5-FU and capecitabine, but was not observed at grades >1 in the present study. This study was small, however, and larger studies would be required to allow definitive conclusions to be drawn regarding the propensity of UFT to cause this syndrome.UFT is a convenient alternative to infusional 5-FU, as it does not require the use of Hickman lines or pumps and therefore the complications associated with these modes of administration can be avoided (Puig-la Calle et al, 1996). Studies have shown that patients prefer UFT to i.v. 5-FU regimens, with most patients citing the convenience of oral treatment as a reason for this preference (Borner et al, 2002; Rocha Lima and del Giglio, 2005). However, patient preference is largely driven by tolerability, as shown by a recent study comparing capecitabine with the Nordic 5-FU/LV regimen (Pfeiffer et al, 2006). In that study, i.v. 5-FU was preferred to oral capecitabine, primarily because capecitabine-treated patients had a higher incidence of adverse events, including diarrhoea and hand–foot syndrome, whereas the Nordic 5-FU regimen was well tolerated.One concern regarding the use of intensive up-front triple therapy is that the choice of second-line treatment may be limited when patients progress. This is not the case with SCOUT, as the tolerability of the alternating regimen meant that most patients (67% of those in the phase I/II study group) were able to receive further treatment, including repeated courses of SCOUT. Re-treatment with SCOUT, which occurred in 33% patients eligible for second-line therapy, resulted in further complete and partial responses. This is in agreement with a previous report that first-line FOLFOXIRI (5-FU, oxaliplatin and irinotecan) did not compromise the feasibility of second-line treatments (Masi et al, 2006). In that study, 76% patients received second-line therapy and further responses were observed in 33% patients, similar to our findings. By virtue of their disease being chemosensitive, most patients (75%) who were suitable for second-line SCOUT went on to receive a third-line regimen, in contrast to only 25% of those who resumed alternate second-line chemotherapy. Again, this lends support to the good tolerability of SCOUT as well as to the concept that further alternate lines of chemotherapy are still possible after SCOUT.Other triple-drug regimens have been shown to result in excellent ORRs and survival. Objective response rates of 43–72% and OS of 21.5–28.4 months have been reported for FOLFOXIRI (Masi et al, 2004; Souglakos et al, 2006; Falcone et al, 2007). Variations in response and survival outcomes are likely to be a result of differences in the regimens used, for example, the doses of irinotecan, oxaliplatin and 5-FU/LV were higher in the study by Falcone and colleagues. In addition, the characteristics of patients entered into the study also differed: patients in the study by Souglakos and colleagues were not required to be younger than 75 years and the proportion of patients with Eastern Cooperative Oncology Group performance status 2 was considerably higher (50% compared with 35% in the Hellenic Group study). The triple-drug combination of capecitabine, oxaliplatin and irinotecan (XELOXIRI) has also been investigated (Masi et al, 2007; Bajetta et al, 2007b). Efficacy outcomes were good, with an ORR of 63% and median OS of 23.5 months in the study by Bajetta and colleages, and an ORR of 70% and median progression-free survival of 9.2 months in the study by Masi and colleagues. FOLFOXIRI and XELOXIRI, however, caused considerable haematological toxicity, neurotoxicity and diarrhoea. Grade 3/4 neutropaenia appears to be a particularly common problem with concurrent triple-drug therapy, although this was rare with the SCOUT regimen (grade 3 neutropaenia occurred in only 3% patients).Others have used 5-FU- and capecitabine-based alternating regimens with good results. Objective response rates of 46–54% and OS of 18–18.7 months have been observed in alternating 5-FU-based regimens, along with little neurotoxicity and myelotoxicity (Aparicio et al, 2005; Ferrari et al, 2005; Hebbar et al, 2006). Cassinello et al (2006) reported a somewhat lower ORR of 37% and OS of 16.4 months for a regimen comprising oxaliplatin on day 1 with capecitabine on days 1–14 of a 21-day cycle, followed by irinotecan on day 1 plus capecitabine on days 1–14 of a second 21-day cycle. Nonetheless, this regimen was well tolerated, providing further support for the improved tolerability of alternating regimens.Considerable debate surrounds the optimal approach to treating patients with mCRC. Results from the CAIRO and FOCUS studies suggest that first-line monotherapy followed by second-line combination therapy is as effective as combination chemotherapy in the first line followed by monotherapy (Koopman et al, 2007; Seymour et al, 2007). In contrast, OPTIMOX1 indicated that intensive first-line therapy with prolonged maintenance treatment and planned reintroduction of intensive therapy is a valid approach to treatment (Tournigand et al, 2006). The OPTIMOX2 study has demonstrated, however, that a break in therapy between intensive treatments cannot be recommended (Maindrault-Goebel et al, 2007). The SCOUT approach avoids the need for discontinuing oxaliplatin, although reducing treatment intensity between full-dose courses may be attractive to some patients.Several limitations of this study need to be considered. This was a single-centre, non-randomised study, making direct comparisons with other studies difficult, and the patient numbers were small. In addition, this study did not include a targeted agent, such as cetuximab or bevacizumab, and therefore the question whether targeted agents might improve response and survival rates remains unanswered. However, it can be argued that the first priority in combination chemotherapy scheduling should be optimisation of the three well-known, conventional, active chemotherapy agents in mCRC. As stated by Saltz at ASCO in 2002, ‘we need all three drugs. We have conflicting data about how to best use them, but, clearly, we need to have them all available to our patients’. Evaluation of the role of the targeted agents combined with a well-tolerated and effective alternating regimen, like SCOUT, would be the next key step to improve outcomes further. Such a study is underway, in which the SCOUT regimen will be combined with cetuximab (E-SCOUT).In conclusion, UFT with LV plus alternating irinotecan and oxaliplatin is an effective, well-tolerated treatment approach for patients with mCRC. SCOUT results in high ORRs (63%, 95% CI: 49–80), respectable survival (median TTP and OS were 8.7 months (95% CI: 7.9–10.4) and 16.8 months (95% CI: 9.6–25.3), respectively), and the feasibility of second- and third-line treatments. 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Tournigand C, Andre T, Achille E, Lledo G, Flesh M, Mery-Mignard D, Quinaux E, Couteau C, Buyse M, Ganem G, Landi B, Colin P, Louvet C, de Gramont A (2004) FOLFIRI followed by FOLFOX6 or the reverse sequence in advanced colorectal cancer: a randomized GERCOR study. J Clin Oncol\n22: 229–23714657227\n38. Tournigand C, Cervantes A, Figer A, Lledo G, Flesch M, Buyse M, Mineur L, Carola E, Etienne PL, Rivera F, Chirivella I, Perez-Staub N, Louvet C, Andre T, Tabah-Fisch I, de Gramont A (2006) OPTIMOX1: a randomized study of FOLFOX4 or FOLFOX7 with oxaliplatin in a stop-and-go fashion in advanced colorectal cancer – a GERCOR study. J Clin Oncol\n24: 394–40016421419\n39. Verso M, Agnelli G (2003) Venous thromboembolism associated with long-term use of central venous catheters in cancer patients. J Clin Oncol\n21: 3665–367514512399"
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+    "id": "PMC2528143",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2528143\nAUTHORS: L Nolan, P W M Johnson, A Ganesan, G Packham, S J Crabb\n\nABSTRACT:\nHistone deacetylase inhibitors have progressed rapidly from the laboratory to clinical testing. This review highlights the promising data for their combination with a wide range of established and novel anticancer agents and discusses the mechanisms that underpin these interactions.\n\nBODY:\nHistone deacetylase inhibitors (HDIs) have progressed rapidly from the laboratory to clinical testing as novel anticancer agents, culminating in the approval of SAHA (Vorinostat, Merck, Whitehouse Station, NJ, USA) for the treatment of recurrent cutaneous T-cell lymphoma. However, despite their promising activity in pre-clinical models, HDIs have demonstrated only modest antitumour activity in initial clinical trials in solid malignancies. In this review, we will discuss current findings to support the hypothesis that in most scenarios, combination with other therapeutic modalities will be required to optimise efficacy and current evidence for the molecular mechanisms that underpin potential combinations.HDIs – structure and mechanism of actionHistone deacetylase (HDAC) enzymes counter the activity of histone acetyltransferases by inducing hydrolysis of the ε-amino acetyl moiety on specific acetylated lysine residues within core histones (Figure 1A and B). Histone acetylation participates in transcriptional regulation in concert with other epigenetic events such as DNA and histone methylation. Histone deacetylase inhibitors induce accumulation of acetylated histones, resulting in the relaxation of chromatin structure and promoting access to transcriptional machinery. Surprisingly, these transcriptional effects are relatively selective, only affecting around 3–10% of the transcriptome, with both the induction and repression of gene expression targets. There may be a common gene expression signature in response to HDIs. For example, reversal of epigenetic silencing of the p21WAF1/CIP1 cyclin-dependent kinase inhibitor is observed in various cancer cell types and for all HDIs. However, cell-specific effects such as modulation of nuclear hormone receptors or cell signalling pathways may vary in biological importance in particular cancer types, which may be relevant for combination strategies.Although the modulation of transcription through histone modification serves as a useful paradigm, the mechanisms that mediate the anticancer effects of HDIs are more complex, with many non-histone targets identified (Figure 2).There are multiple HDIs in clinical development and key representatives are shown in Figure 1C. These molecules are structurally diverse, but with the common feature of employing a chemical ‘war-head’ active site that chelates the zinc atom in the active site of Class I and/or II HDACs, thereby blocking enzyme activity. With the exception of very simple aliphatic acids such as valproic acid (VPA), these molecules conform to a common pharmacophore (Figure 1C), comprising a ‘linker’ mimicking a lysine side chain, and a ‘cap’ structure of variable size that makes additional interactions around the rim of the enzyme active site.HDI combinations with cytotoxic chemotherapyIn an attempt to find a niche for HDIs, they have been tested in combination with a variety of conventional cytotoxic chemotherapeutic agents. Pre-clinical data in multiple cancer cell lines (including breast, ovarian, pancreatic, colon, non-small cell lung, prostate, thyroid, hepatocellular and oral squamous cell carcinomas and melanoma) have shown the potentiation by HDIs of the effects of topoisomerase I inhibitors (camptothecin, irinotecan, topotecan) topoisomerase II inhibitors (epirubicin, doxorubicin, etoposide, mitoxantrone) and other DNA-damaging agents (cisplatin, oxaliplatin, bleomycin). In vitro, synergistic induction of apoptosis is seen when HDIs are combined with epirubicin in breast cancer cells and with etoposide or cisplatin in melanoma cells (Marchion et al, 2005; Valentini et al, 2007). In vivo potentiation of epirubicin has been confirmed in breast cancer xenograft models (Marchion et al, 2005). Subsequently, a phase I clinical trial of VPA combined with epirubicin showed a 22% partial response rate in multiply pretreated solid malignancies (Munster et al, 2007). It is worth noting that these investigators began VPA 48 h before infusion of epirubicin on each cycle to attempt to exploit synergism seen with this sequencing approach demonstrated in pre-clinical models (see below). Dose-limiting toxicity was predominantly neurovestibular (including dizziness, confusion and hearing loss) or gastrointestinal (diarrhoea). Anthracycline-induced toxicity in this trial did not appear to be worsened by combination with VPA, and other toxicities are manageable providing some reassurance that any synergy in terms of efficacy will not be replicated in unwanted side effects. A phase II trial combining VPA with chemotherapy (5FU, epirubicin and cyclophosphamide) is currently recruiting patients with metastatic breast cancer (www.clinicaltrials.gov).A number of mechanisms may account for the potentiation of DNA-damaging agents by HDIs, reflecting their pleiotropic actions. For topoisomerase inhibitors, HDAC1 and -2 have been shown to bind and interact with topoisomerase II, and form an integral part of the NuRD complex (Tsai et al, 2000). Scheduling appears to be critical in explaining the variable potentiating effects of HDIs on topoisomerase II inhibitors, with pre-exposure of breast cancer cells to vorinostat for 48 h needed to induce synergistic apoptosis, increase nuclear epirubicin levels and increase DNA damage. Shorter pre-exposure periods abrogated synergy, and exposure after chemotherapy resulted in antagonistic effects, implying that HDI relaxation of chromatin allows greater access for topoisomerase II inhibition, but possibly also stabilises the topoisomerase II–DNA complex further resulting in more efficient generation of strand breaks. Expression of the chemotherapeutic target may also be crucial as potentiation was lost in topoisomerase II null cells if the HDI was combined with epirubicin but not topotecan (Marchion et al, 2004).Sequencing was also important for combination with topoisomerase I inhibition but with apparent benefit for HDI exposure after the chemotherapeutic to exploit cell cycle effects of each agent. Potentiation has been shown for an HDI added 24–48 h after camptothecin in breast and lung cancer cells. Cells arrested in G2-M by camptothecin appeared most sensitive to subsequent HDI addition possibly through HDI-induced decreases in cyclin B levels and of the antiapoptotic proteins XIAP and survivin. These findings suggested that reduced expression of these antiapoptotic factors could increase efficacy of topoisomerase I inhibitors if given in a sequence that does not prevent tumour cell progression through S phase (Bevins and Zimmer, 2005). Enhancement of cisplatin-induced apoptosis by HDIs in oral squamous cell carcinoma has also been shown to be greater if the HDI is given concurrently or following chemotherapy rather than prior. Experiments suggested that cells arrested at the G1/S checkpoint by cisplatin were more sensitive to HDAC inhibition through enhancement of reactive oxygen species generation and caspase-3 activation. Histone deacetylase inhibitor therapy decreased intracellular reduced glutathione. Thus, HDIs appeared to disrupt intracellular redox balance, inducing maximal apoptosis at G1/S arrest and potentiating platinum response (Sato et al, 2006). Taken together, there is mounting pre-clinical evidence that HDIs synergistically potentiate chemotherapeutic agents that exploit topoisomerase enzymes and DNA damage. Clinical investigation now in progress will elucidate whether these promising findings translate to the clinic and also if interactions impact on toxicity (Table 1).Taxanes, which inhibit microtubule depolymerisation during metaphase resulting in increased microtubule formation and activation of mitosis checkpoints leading to apoptosis, have also been investigated in combination with HDIs. Synergistic reductions in growth were seen in endometrial cancer cells following treatment with paclitaxel combined with the HDI trichostatin A (TSA), and this was confirmed in mouse xenograft studies (Dowdy et al, 2006). Synergistic interaction was also seen in breast cancer cells combining docetaxel with vorinostat (Bali et al, 2005). There are no published clinical data for HDI–taxane combinations but trials are ongoing in breast and gynaecologic cancers (Table 1). With regard to underlying mechanisms, in endometrial cancer cell lines, TSA administration induced α-tubulin acetylation and appeared to stabilise microtubules. Combination with paclitaxel led to a significant increase in acetylated tubulin and microtubule stabilisation above that with either agent alone (Dowdy et al, 2006).These data show a clear rationale for combining HDIs with a range of chemotherapeutic agents, but that clinical trials must be underpinned by a clear mechanistic rationale specific to both experimental agents and tumour types.HDI combinations with agents targeting the human epidermal growth factor receptor (HER) familyTargeted therapies aimed at the HER family have advanced treatment of a range of common malignancies including breast, colorectal and lung cancers. The main data regarding HDI combinations to optimise this approach are for HER2-overexpressing breast cancer. Trastuzumab, a humanised monoclonal antibody to the HER2 extra cellular domain, is effective for those with receptor overexpression; however, optimisation of HER2-targeted therapy and avoidance of resistance mechanisms are required (Crabb and Chia, 2007). In vitro studies indicate that HDIs have single-agent activity in HER2-overexpressing breast cancer cell lines including attenuation of HER2 expression, its tyrosine kinase activity, its cell membrane localisation and dimerisation with HER3 (Fuino et al, 2003; Bali et al, 2005). Combination with trastuzumab produced synergistic induction of apoptosis (Fuino et al, 2003; Bali et al, 2005). Synergy may result from counteracting HER2 overexpression as HDAC inhibition reduced HER2 mRNA transcript expression and induced HER2 protein degradation (Scott et al, 2002; Fuino et al, 2003). The latter mechanism occurred through the acetylation of heat shock protein (HSP)90, causing its inactivation and loss of multiple HSP90 client proteins. HSP90 acetylation decreased ATP binding, inducing a shift from HER2 binding with HSP90 to HSP70, resulting in HER2 targeting for ubiquitination and proteasomal degradation (Fuino et al, 2003). On the basis of these in vitro data, clinical trials of trastuzumab–HDI combinations are in progress for locally advanced and metastatic breast cancer.It remains unproven to what degree synergism between HDIs and trastuzumab in HER2-positive breast cancer models might also apply to other HER2-directed therapeutics that are in various stages of clinical testing (Crabb and Chia, 2007). However, inhibition of proliferation, apoptosis and signalling inhibition were potentiated when vorinostat was co-administered with the pan-HER tyrosine kinase inhibitor CI-1033 in breast as well as prostate and head and neck squamous carcinoma cells.Regarding other members of the HER family, in non-small cell lung cancer (NSCLC), synergy has been shown between HDIs and the HER1 (EGFR) tyrosine kinase inhibitors erlotinib and gefitinib (Witta et al, 2006). Histone deacetylases are recruited by transcriptional repressors such as Slug/Snail and ZEB1, which are implicated in resistance mechanisms to these agents, and gefitinib sensitivity appeared to be restored in NSCLC cell line models of gefitinib resistance when combined with an HDI (Witta et al, 2006). In a separate study, HSP90 acetylation and reduced association with HER1, Akt and STAT3 were seen in cell lines harbouring HER1 kinase mutations following exposure to HDIs leading to apoptosis. Conversely, little effect on apoptosis was seen in cells not dependent on HER1 through kinase mutations. Therefore, HER1 mutation status might be a predictive factor for HDI combination with HER1 inhibitors in this setting. This highlights the value in dissecting the mechanisms underlying beneficial combinations to allow for rational targeting of appropriate cancer phenotypes early in clinical development.HDI combination with proteasome inhibitionProteasome inhibitors act by binding within the catalytic 20S core of the proteasome, resulting in the build-up of proteins targeted for degradation. Cancer cells appear more likely to accumulate misfolded proteins than normal cells, producing a therapeutic window with promising activity in haematological malignancies. Other evidences exist regarding synergistic interactions with HDIs. Treating myeloma cell lines with bortezomib followed by vorinostat produced synergistic induction of mitochondrial injury, caspase activation and apoptosis associated with NF-κB inactivation (Pei et al, 2004). Similar findings were also observed in BCR/ABL-positive and -negative leukaemia cell lines and, interestingly, in solid tumour cell lines.Combined HDAC–proteasomal inhibition may be effective because both interact with NF-κB pathways. Proteasome inhibitors cause accumulation of IκBα increasing its NF-κB binding, thereby preventing nuclear localisation and activation of NF-κB target genes. NF-κB subunits are acetylated at multiple lysine residues by p300/CBP acetyltransferases. Acetylation of different residues regulates different NF-κB functions (including transcriptional activation, DNA-binding affinity, IκBα assembly and subcellular localisation), and HDIs manipulate gene expression patterns resulting from NF-κB activation both directly through NF-κB subunit acetylation and indirectly through chromatin remodelling (Chen and Greene, 2003; Graham and Gibson, 2005).HDI combination with hormonal therapyAnother combination approach for targeting aberrant gene silencing directly is with retinoic acid in acute promyelocytic leukaemia. Acute promyelocytic leukaemia is characterised by translocations of the retinoic acid receptor A (RARA) gene most commonly with the PML gene. The resulting fusion protein transcription factor has enhanced co-repressor-binding properties, increasing HDAC and DNA methyltransferase recruitment. This aberrant retinoid signalling results in potent transcriptional silencing of target genes. All trans-retinoic acid (ATRA), alone or combined with chemotherapy, is effective in reversing this silencing but resistance may occur. Addition of an HDI to this combination is logical in view of the enhanced co-repressor binding and HDAC recruitment by RARA fusion proteins. Histone deacetylase inhibitor therapy alone does not induce differentiation in APL but can induce this in retinoic acid-resistant cell lines when the two agents are combined (Altucci et al, 2005) In APL, this approach requires clinical testing to prove its effectiveness over retinoic acid treatment alone. In acute myeloid leukaemia (AML) or myelodysplastic syndrome (MDS) either unsuitable for or relapsed following conventional therapy, combined VPA and ATRA has produced modest efficacy in phase I/II clinical trials with apparently manageable toxicity. It remains unclear, from these studies, to what extent the combination adds to either drug given as monotherapy, and further data in this area would be of interest (Bug et al, 2005; Kuendgen et al, 2005, 2006).Histone deacetylase inhibitors may be of value in combination with hormonal therapy for breast cancer. They potentiate the antiproliferative effects of the selective oestrogen receptor (ER) modulators tamoxifen and raloxifene, the pure antioestrogen fulvestrant and the aromatase inhibitor letrozole in breast cancer cell lines. Interestingly, the partial agonistic effect of endometrial adenocarcinoma cell proliferation induced by tamoxifen was blocked by HDI co-administration (Hodges-Gallagher et al, 2007). In a further work, treatment with an HDI rendered formerly unresponsive ERα-negative breast cancer cells responsive to tamoxifen. HDI enhanced overall ER transcriptional activity in these cells. Interestingly, this appeared to be by inducing the expression and nuclear translocation of ERβ but not ERα. Reduction of ERβ expression by short interfering RNA abrogated this HDI-induced sensitisation effect (Jang et al, 2004). Evidence exists to suggest that DNA methylation and histone deacetylation interact to maintain a repressive chromatin complex at the ER promoter. Inhibition of either may be sufficient to activate the silenced ER gene (Zhou et al, 2007). Clinical investigation following these observations is in progress.HDI combination with targeted agents for BCR/ABL-positive leukaemiaThe role of HDIs in combination with agents targeting the BCR/ABL oncoprotein has been investigated in chronic myeloid leukaemia (CML) for which the BCR/ABL tyrosine kinase inhibitor imatinib is now a standard therapy but resistance can develop. In vitro, vorinostat has been shown to synergistically enhance the activity of imatinib and the second-generation agent dasatinib possibly through the inhibition of HSP90 chaperone function. In addition to downregulation of BCR/ABL expression, multiple perturbations in signalling and cell cycle-regulatory proteins are induced by this combination including the Ras/Raf/MEK/ERK, Akt, STAT and JNK pathways and cyclin D1 (Yu et al, 2003). Subsequently, combination of HDI with sorafenib, an inhibitor of multiple kinases including Raf-1, platelet-derived growth factor, vascular endothelial growth factor receptors 1 and 2, and FLT3, was found to induce synergistic cell death in BCR/ABL-positive cells, imatinib-resistant cells and primary CD34+ bone marrow cells from CML patients (Dasmahapatra et al, 2007).Interestingly, both imatinib and sorafenib blocked the HDI-mediated induction of p21WAF1/CIP1, perhaps the most consistently observed HDI downstream molecular effect. Forced expression of p21WAF1/CIP1 depleted the combined HDI/sorafenib effect, implying that this may be required for synergism. Potential mechanisms include disruption of p21WAF1/CIP1-mediated G1 arrest, interference with its direct antiapoptotic actions such as inhibition of caspase-3 or c-Jun NH2-terminal kinase activation or disruption of the upstream Raf/MEK/ERK axis (Yu et al, 2003; Dasmahapatra et al, 2007).HDIs in combination with other epigenetic modifiersOne reason for the relatively modest number of genes affected by HDIs is the dominant effect of methylation status over acetylation. Dual administration of HDIs with DNA-hypomethylating agents is therefore of interest. Aberrant DNA methylation is characteristic of a number of myeloid leukaemias (Garcia-Manero et al, 2002) and dual epigenetic modulation might allow suppression of the malignant clone. In vitro, the combination of VPA and 5-aza-2′-deoxycytidine (decitabine) produced synergistic growth inhibition and induction of apoptosis in leukaemia cell lines (Yang et al, 2005). In a subsequent phase I/II clinical trial of this combination, 54 patients with AML or high-risk MDS, either relapsed or unsuitable for first-line chemotherapy, received a fixed dose of decitabine and escalating doses of VPA. Twelve patients (22%) had objective responses with 10 complete remissions. Major cytogenetic response was documented in six of eight responders. Of the five target genes investigated, hypomethylation of the key cell cycle-regulating gene, p15 was found to be the best indicator of response. Pretreatment p15 methylation was significantly lower in responders vs non-responders; however, neither the absolute or the percentage change in p15 methylation was statistically significant, and responses were not correlated with the induction of H3 or H4 acetylation (Garcia-Manero et al, 2006). In another study, utilising phenylbutyrate and 5-azacytidine in a similar patient population, 11 of 29 patients responded. Furthermore, six of six with pretreatment methylation of p15 or CDH-1 (E-cadherin) promoters reversed methylation during the first cycle of therapy whereas none of the six non-responders showed any demethylation (Gore et al, 2006). Further identification and validation of predictive biomarkers would be of immense value in the ongoing clinical assessment of HDIs in this and other settings but may need to be tumour- and combination-specific. Toxicity in these clinical studies included neurological (encephalopathy, confusion, somnolence) and haematological (thrombocytopaenia, neutropaenia) events and fatigue, but the combinations were considered well tolerated. Further clinical evaluation will be required to establish the value of combination therapy over monotherapy approaches and compared to conventional chemotherapeutic interventions.HDI combination with ionising radiationHistone deacetylase inhibitors can act as radiosensitisers (Karagiannis and El-Osta, 2006). For example, in NSCLC cells, synergism has been demonstrated between HDIs and irradiation for induction of apoptosis and inhibition of clonogenic survival and confirmed in in vivo tumour studies. Following irradiation, γ-H2AX foci following irradiation, a conserved response to DNA double-strand break formation necessary for recruitment of many factors involved in DNA repair was found to be increased by combination with an HDI. Furthermore, radiation alone induced translocation of HDAC4 to the nucleus whereas combination therapy resulted in its confinement to the cytoplasm (Geng et al, 2006). The DNA damage-sensing protein, 53BP1, has been shown to co-localise to the nucleus with HDAC4 in response to double-strand DNA breaks (Kao et al, 2003). Therefore, HDI therapy may potentiate radiation in part by the suppression of HDAC incorporation into DNA damage-signalling and -repair complexes. Other factors are also likely to be relevant. For example, following DNA damage, checkpoint molecules activate ataxia telangiectasia-mutated protein (ATM), which in turn phosphorylates effectors. Histone deacetylase-1 is known to interact with ATM and this interaction is enhanced by ionising radiation and inhibited by HDAC inhibition (Kim et al, 1999). Histone deacetylases are also important for the repair of established double-strand breaks, with the expression of Ku70, Ku86 and other repair proteins decreased by HDI therapy despite radiation-induced DNA damage (Munshi et al, 2005). These pre-clinical studies demonstrate the range of potential mechanisms that are implicated in HDI-mediated radiosensitisation and a variety of clinical studies are in progress (Table 1).ConclusionsThe niche for HDIs in the treatment of cancer remains inadequately defined. Understanding of the diverse mechanisms for their anticancer action continues to increase, including molecular mechanisms elucidated by studying combination therapy. In the face of modest activity as single agents, except in cutaneous T-cell lymphoma (CTCL) where the unique tumour microenvironment may account for their unpredicted efficacy, their ability to synergise with, and potentially overcome resistance to, many other agents represents a promising strategy for clinical development. Current evidence to support this assertion is predominantly pre-clinical, with only a small number of non-randomised early-phase clinical trials reported (of combinations with anthracyclines, ATRA or DNA-hypomethylating agents). We therefore wait for clear proof that the multiple promising combinations tested in pre-clinical studies can in fact translate to added clinical value for patients above use of single or other agents. If this can be achieved, then the next few years should herald clinical data from phase II and III studies to define the exact value of combination approaches and improved understanding of mechanisms that underpin activity.\n\nREFERENCES:\n1. Altucci L, Clarke N, Nebbioso A, Scognamiglio A, Gronemeyer H (2005) Acute myeloid leukemia: therapeutic impact of epigenetic drugs. Int J Biochem Cell Biol\n37: 1752–176215964234\n2. Bali P, Pranpat M, Swaby R, Fiskus W, Yamaguchi H, Balasis M, Rocha K, Wang HG, Richon V, Bhalla K (2005) Activity of suberoylanilide hydroxamic acid against human breast cancer cells with amplification of her-2. Clin Cancer Res\n11: 6382–638916144943\n3. Bevins RL, Zimmer SG (2005) It's about time: scheduling alters effect of histone deacetylase inhibitors on camptothecin-treated cells. Cancer Res\n65(15): 6957–696616061681\n4. Bug G, Ritter M, Wassmann B, Schoch C, Heinzel T, Schwarz K, Romanski A, Kramer OH, Kampfmann M, Hoelzer D, Neubauer A, Ruthardt M, Ottmann OG (2005) Clinical trial of valproic acid and all-trans retinoic acid in patients with poor-risk acute myeloid leukemia. Cancer\n104: 2717–272516294345\n5. Chen LF, Greene WC (2003) Regulation of distinct biological activities of the NF-kappaB transcription factor complex by acetylation. J Mol Med\n81: 549–55712920522\n6. Crabb SJ, Chia SK (2007) HER2 directed therapies. Adv Breast Cancer\n4: 40–47\n7. Dasmahapatra G, Yerram N, Dai Y, Dent P, Grant S (2007) Synergistic interactions between vorinostat and sorafenib in chronic myelogenous leukemia cells involve Mcl-1 and p21CIP1 down-regulation. Clin Cancer Res\n13: 4280–429017634558\n8. Dowdy SC, Jiang S, Zhou XC, Hou X, Jin F, Podratz KC, Jiang SW (2006) Histone deacetylase inhibitors and paclitaxel cause synergistic effects on apoptosis and microtubule stabilization in papillary serous endometrial cancer cells. Mol Cancer Ther\n5: 2767–277617121923\n9. Fuino L, Bali P, Wittmann S, Donapaty S, Guo F, Yamaguchi H, Wang HG, Atadja P, Bhalla K (2003) Histone deacetylase inhibitor LAQ824 down-regulates Her-2 and sensitizes human breast cancer cells to trastuzumab, taxotere, gemcitabine, and epothilone B. Mol Cancer Ther\n2: 971–98414578462\n10. Garcia-Manero G, Daniel J, Smith TL, Kornblau SM, Lee MS, Kantarjian HM, Issa JP (2002) DNA methylation of multiple promoter-associated CpG islands in adult acute lymphocytic leukemia. Clin Cancer Res\n8: 2217–222412114423\n11. Garcia-Manero G, Kantarjian HM, Sanchez-Gonzalez B, Yang H, Rosner G, Verstovsek S, Rytting M, Wierda WG, Ravandi F, Koller C, Xiao L, Faderl S, Estrov Z, Cortes J, O'Brien S, Estey E, Bueso-Ramos C, Fiorentino J, Jabbour E, Issa JP (2006) Phase 1/2 study of the combination of 5-aza-2′-deoxycytidine with valproic acid in patients with leukemia. Blood\n108: 3271–327916882711\n12. Geng L, Cuneo KC, Fu A, Tu T, Atadja PW, Hallahan DE (2006) Histone deacetylase (HDAC) inhibitor LBH589 increases duration of {gamma}-H2AX foci and confines HDAC4 to the cytoplasm in irradiated non-small cell lung cancer. Cancer Res\n66: 11298–1130417145876\n13. Gore SD, Baylin S, Sugar E, Carraway H, Miller CB, Carducci M, Grever M, Galm O, Dauses T, Karp JE, Rudek MA, Zhao M, Smith BD, Manning J, Jiemjit A, Dover G, Mays A, Zwiebel J, Murgo A, Weng LJ, Herman JG (2006) Combined DNA methyltransferase and histone deacetylase inhibition in the treatment of myeloid neoplasms. Cancer Res\n66: 6361–636916778214\n14. Graham B, Gibson SB (2005) The two faces of NFkappaB in cell survival responses. Cell Cycle\n4: 1342–134516123594\n15. Hodges-Gallagher L, Valentine CD, Bader SE, Kushner PJ (2007) Inhibition of histone deacetylase enhances the anti-proliferative action of antiestrogens on breast cancer cells and blocks tamoxifen-induced proliferation of uterine cells. Breast Cancer Res Treat\n105: 297–30917186358\n16. Jang ER, Lim SJ, Lee ES, Jeong G, Kim TY, Bang YJ, Lee JS (2004) The histone deacetylase inhibitor trichostatin A sensitizes estrogen receptor alpha-negative breast cancer cells to tamoxifen. Oncogene\n23: 1724–173614676837\n17. Kao GD, McKenna WG, Guenther MG, Muschel RJ, Lazar MA, Yen TJ (2003) Histone deacetylase 4 interacts with 53BP1 to mediate the DNA damage response. J Cell Biol\n160: 1017–102712668657\n18. Karagiannis TC, El-Osta A (2006) Modulation of cellular radiation responses by histone deacetylase inhibitors. Oncogene\n25: 3885–389316462761\n19. Kim GD, Choi YH, Dimtchev A, Jeong SJ, Dritschilo A, Jung M (1999) Sensing of ionizing radiation-induced DNA damage by ATM through interaction with histone deacetylase. J Biol Chem\n274: 31127–3113010531300\n20. Kuendgen A, Knipp S, Fox F, Strupp C, Hildebrandt B, Steidl C, Germing U, Haas R, Gattermann N (2005) Results of a phase 2 study of valproic acid alone or in combination with all-trans retinoic acid in 75 patients with myelodysplastic syndrome and relapsed or refractory acute myeloid leukemia. Ann Hematol\n84(Suppl 1): 61–66\n21. Kuendgen A, Schmid M, Schlenk R, Knipp S, Hildebrandt B, Steidl C, Germing U, Haas R, Dohner H, Gattermann N (2006) The histone deacetylase (HDAC) inhibitor valproic acid as monotherapy or in combination with all-trans retinoic acid in patients with acute myeloid leukemia. Cancer\n106: 112–11916323176\n22. Marchion DC, Bicaku E, Daud AI, Richon V, Sullivan DM, Munster PN (2004) Sequence-specific potentiation of topoisomerase II inhibitors by the histone deacetylase inhibitor suberoylanilide hydroxamic acid. J Cell Biochem\n92: 223–23715108350\n23. Marchion DC, Bicaku E, Daud AI, Sullivan DM, Munster PN (2005) Valproic acid alters chromatin structure by regulation of chromatin modulation proteins. Cancer Res\n65: 3815–382215867379\n24. Munshi A, Kurland JF, Nishikawa T, Tanaka T, Hobbs ML, Tucker SL, Ismail S, Stevens C, Meyn RE (2005) Histone deacetylase inhibitors radiosensitize human melanoma cells by suppressing DNA repair activity. Clin Cancer Res\n11: 4912–492216000590\n25. Munster P, Marchion D, Bicaku E, Schmitt M, Lee JH, DeConti R, Simon G, Fishman M, Minton S, Garrett C, Chiappori A, Lush R, Sullivan D, Daud A (2007) Phase I trial of histone deacetylase inhibition by valproic acid followed by the topoisomerase II inhibitor epirubicin in advanced solid tumors: a clinical and translational study. J Clin Oncol\n25: 1979–198517513804\n26. Pei X-Y, Dai Y, Grant S (2004) Synergistic induction of oxidative injury and apoptosis in human multiple myeloma cells by the proteasome inhibitor bortezomib and histone deacetylase inhibitors. Clin Cancer Res\n10: 3839–385215173093\n27. Sato T, Suzuki M, Sato Y, Echigo S, Rikiishi H (2006) Sequence-dependent interaction between cisplatin and histone deacetylase inhibitors in human oral squamous cell carcinoma cells. Int J Oncol\n28: 1233–124116596240\n28. Scott GK, Marden C, Xu F, Kirk L, Benz CC (2002) Transcriptional repression of ErbB2 by histone deacetylase inhibitors detected by a genomically integrated ErbB2 promoter-reporting cell screen. Mol Cancer Ther\n1: 385–39212477051\n29. Tsai SC, Valkov N, Yang WM, Gump J, Sullivan D, Seto E (2000) Histone deacetylase interacts directly with DNA topoisomerase II. Nat Genet\n26: 349–35311062478\n30. Valentini A, Gravina P, Federici G, Bernardini S (2007) Valproic acid induces apoptosis, p16INK4A upregulation and sensitization to chemotherapy in human melanoma cells. Cancer Biol Ther\n6: 185–19117218782\n31. Witta SE, Gemmill RM, Hirsch FR, Coldren CD, Hedman K, Ravdel L, Helfrich B, Dziadziuszko R, Chan DC, Sugita M, Chan Z, Baron A, Franklin W, Drabkin HA, Girard L, Gazdar AF, Minna JD, Bunn Jr PA (2006) Restoring E-cadherin expression increases sensitivity to epidermal growth factor receptor inhibitors in lung cancer cell lines. Cancer Res\n66: 944–95016424029\n33. Yang H, Hoshino K, Sanchez-Gonzalez B, Kantarjian H, Garcia-Manero G (2005) Antileukemia activity of the combination of 5-aza-2′-deoxycytidine with valproic acid. Leuk Res\n29: 739–74815927669\n34. Yu C, Rahmani M, Almenara J, Subler M, Krystal G, Conrad D, Varticovski L, Dent P, Grant S (2003) Histone deacetylase inhibitors promote STI571-mediated apoptosis in STI571-sensitive and -resistant Bcr/Abl+ human myeloid leukemia cells. Cancer Res\n63: 2118–212612727828\n35. Zhou Q, Atadja P, Davidson NE (2007) Histone deacetylase inhibitor LBH589 reactivates silenced estrogen receptor alpha (ER) gene expression without loss of DNA hypermethylation. Cancer Biol Ther\n6: 64–6917172825"
+}

batch_11/PMC2528147.json

@@ -0,0 +1,4 @@
+{
+    "id": "PMC2528147",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2528147\nAUTHORS: R D Blumenthal, H J Hansen, D M Goldenberg\n\nABSTRACT:\nNo Abstract\n\nBODY:\nSir,\nElevated expression of carcinoembryonic antigen (CEA; CD66e; CEACAM5) has been implicated in various biological aspects of neoplasia. Carcinoembryonic antigen has been shown to be involved in both homophilic and heterophilic binding (Benchimol et al, 1989), suggesting to some that it is an intercellular adhesion molecule involved in cancer invasion, adhesion, and metastasis (Yoshioka et al, 1998). Carcinoembryonic antigen also serves an anti-apoptotic function (Ordonez et al, 2000). Studies have shown that CEA affects the expression of various groups of cancer-related genes, especially cell cycle and apoptotic genes, protecting colonic tumour cells from various apoptotic stimuli, including 5-FU therapy (Soeth et al, 2001).Conaghan et al, in the March issue of British Journal of Cancer, described in vitro studies with a humanised anti-CEA antibody (PR1A3), demonstrating its ability to react with a panel of CEA-expressing colorectal cancer cell lines and induce ADCC activity. The authors state that ‘there are so far no unconjugated, or ‘naked’ antibodies to CEA being used for treatment of colorectal cancer’ (Conaghan et al, 2008). Since this is not correct, we offer this clarification. Dr Conaghan appears to be unaware of our work in the field of anti-CEA therapy with the MN-14 (labetuzumab) antibody. We have been studying the humanised anti-CEA monoclonal antibody (MAb), hMN-14 or labetuzimab, both prelinically and in patients, for a number of years. This MAb binds with high affinity to the restricted A3B3 domain (Gold group 3) found on CEA (Sharkey et al, 1995). Clinically, the MN-14 antibody was shown to have excellent targeting properties and the potential for reduced immunogenicity (Sharkey et al, 1995), and has been studied as a naked MAb in colorectal and breast cancer patients (Immunomedics Inc., unpublished results), as well as a radioconjugate (Hajjar et al, 2002; Liersch et al, 2005). The technology to produce large quantities of recombinant humanised MN-14 antibody in a bioreactor has been described (Qu et al, 2005). Furthermore, chimeric human T cells have been created that express humanised MN-14 Fab, and MN-14 scFv joined to immunoglobulin–T-cell receptors. These ‘designer T cells’ effectively kill CEA-expressing cancer cells, even in the presence of high level of soluble CEA (Nolan et al, 1999).Labetuzumab, as an IgG1 MAb, induces effector-cell function (ADCC) in vitro against CEA-positive human colonic tumour cells (Blumenthal et al, 2005b). Antibody targeting of CEA may also modulate migration, invasion, and adhesion of human cancer cells in vitro (Blumenthal et al, 2005a). Labetuzumab is able to inhibit the growth of lung metastasis from colorectal cancer cells expressing high levels of CEA or from colorectal cancer cells with the lower expression of CEA, but in mice where levels of peripheral WBCs were elevated by G-CSF (Blumenthal et al, 2005b). The MAb also shows chemosensitising properties in both s.c. and metastatic human colonic tumour cells propagated in nude mice. Administration of labetuzumab enhanced the therapeutic effects of 5-FU and CPT-11, two cytotoxic drugs frequently used in colorectal cancer therapy (Blumenthal et al, 2005b). In another high CEA-expressing human medullary thyroid cancer xenograft (TT), we have shown that hMN-14 anti-CEA IgG can inhibit tumour cell growth and also augment the effects of dacarbazine, which is active in this cancer type (Stein and Goldenberg, 2004).Although the PR1A3 antibody described by Conaghan et al in March 2008 demonstrates some promising in vitro activity, the experience with such humanised anti-CEA MAbs goes well beyond these observations, as described by our own in vitro and preclinical studies demonstrating direct, specific, anti-tumour effects without conjugation to a cytotoxic agent, in both colorectal and medullary thryroid cancer xenografts, providing evidence for the superiority of the combined modality naked anti-CEA MAb immunotherapy and chemotherapy treatments. The results support further studies focused on the integration of anti-CEA MAb therapy into chemotherapeutic regimens for the improved management CEA-expressing neoplasms.\n\nREFERENCES:\n1. Benchimol S, Fuks A, Jothy S, Beauchemin N, Shirota K, Stanners CP (1989) Carcinoembryonic antigen, a human tumor marker, functions as an intercellular adhesion molecule. Cell\n57: 327–3342702691\n2. Blumenthal RD, Hansen HJ, Goldenberg DM (2005a) Inhibition of adhesion, invasion, and metastasis by antibodies targeting CEACAM6 (NCA-90) and CEACAM5 (carcinoembryonic antigen). Cancer Res\n65: 8809–881716204051\n3. Blumenthal RD, Osorio L, Hayes MK, Horak ID, Hansen HJ, Goldenberg DM (2005b) Carcinoembryonic antigen antibody inhibits lung metastasis and augments chemotherapy in a human colonic carcinoma xenograft. Cancer Immunol Immunother\n54: 315–32715592930\n4. Conaghan P, Ashraf S, Tytherleigh M, Wilding J, Tchilian E, Bicknell D, Mortensen NJ, Bodmer W (2008) Targeted killing of colorectal cancer cell lines by a humanised IgG1 monoclonal antibody that binds to membrane-bound carcinoembryonic antigen. Br J Cancer\n98: 1217–122518349843\n5. Hajjar G, Sharkey RM, Burton J, Zhang CH, Yeldell D, Matthies A, Alavi A, Losman MJ, Brenner A, Goldenberg DM (2002) Phase I radioimmunotherapy trial with iodine-131–labeled humanized MN-14 anti-carcinoembryonic antigen monoclonal antibody in patients with metastatic gastrointestinal and colorectal cancer. Clin Colorectal Cancer\n2: 31–4212453334\n6. Liersch T, Meller J, Kulle B, Behr TM, Markus P, Langer C, Ghadimi BM, Wegener WA, Kovacs J, Horak ID, Becker H, Goldenberg DM (2005) Phase II trial of carcinoembryonic antigen radioimmunotherapy with 131I-labetuzumab after salvage resection of colorectal metastases in the liver: five-year safety and efficacy results. J Clin Oncol\n23: 6763–677016170184\n7. Nolan KF, Yun CO, Akamatsu Y, Murphy JC, Leung SO, Beecham EJ, Junghans RP (1999) Bypassing immunization: optimized design of ‘designer T cells’ against carcinoembryonic antigen (CEA)-expressing tumors, and lack of suppression by soluble CEA. Clin Cancer Res\n5: 3928–394110632322\n8. Ordonez C, Screaton RA, Ilantzis C, Stanners CP (2000) Human carcinoembryonic antigen functions as a general inhibitor of anoikis. Cancer Res\n60: 3419–342410910050\n9. Qu Z, Griffiths GL, Wegener WA, Chang CH, Govindan SV, Horak ID, Hansen HJ, Goldenberg DM (2005) Development of humanized antibodies as cancer therapeutics. Methods\n36: 84–9515848077\n10. Sharkey RM, Juweid M, Shevitz J, Behr T, Dunn R, Swayne LC, Wong GY, Blumenthal RD, Griffiths GL, Siegel JA, Leung S, Hansen HJ, Goldenberg DM (1995) Evaluation of a complementarity-determining region-grafted (humanized) anti-carcinoembryonic antigen monoclonal antibody in preclinical and clinical studies. Cancer Res\n55: 5935s–55945s7493374\n11. Soeth E, Wirth T, List HJ, Kumbhani S, Petersen A, Neumaier M, Czubayko F, Juhl H (2001) Controlled ribozyme targeting demonstrates an antiapoptotic effect of carcinoembryonic antigen in HT29 colon cancer cells. Clin Cancer Res\n7: 2022–203011448920\n12. Stein R, Goldenberg DM (2004) A humanized monoclonal antibody to carcinoembryonic antigen, labetuzumab, inhibits tumor growth and sensitizes human medullary thyroid cancer xenografts to dacarbazine chemotherapy. Mol Cancer Ther\n3: 1559–156415634649\n13. Yoshioka T, Masuko T, Kotanagi H, Aizawa O, Saito Y, Nakazato H, Koyama K, Hashimoto Y (1998) Homotypic adhesion through carcinoembryonic antigen plays a role in hepatic metastasis development. Jpn J Cancer Res\n89: 177–1859548445"
+}

batch_11/PMC2528156.json

@@ -0,0 +1,4 @@
+{
+    "id": "PMC2528156",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2528156\nAUTHORS: S Q Ashraf, P J Conaghan, J L Wilding, W F Bodmer\n\nABSTRACT:\nNo Abstract\n\nBODY:\nSir,\nIn response to Blumenthal et al, we would like to further clarify a few points that have been raised. The main one relates to the statement made in our paper that ‘there are so far no unconjugated or ‘naked’ antibodies to carcinoembryonic antigen (CEA) being used for the treatment of colorectal cancer’ (Conaghan et al, 2008). Blumenthal et al suggest that this is not correct; however, they fail to provide evidence to the contrary in their letter. They quote three published articles in support of their argument. However, the first one relates to a targeting study using MN-14 (Sharkey et al, 1995). In the 1980s, radiolabelled murine PR1A3 was also demonstrated to be highly specific for human colorectal lesions (Granowska et al, 1989). The other two papers present data relating to the use of a radioconjugate form of MN-14 (Hajjar et al, 2002; Liersch et al, 2005). In the letter from Blumenthal et al, there is reference to unpublished results with unconjugated MN-14 being used in patients. It is however difficult to make an informed response regarding unpublished data. Thus, to our knowledge, the point made in our paper still holds true: no unconjugated antibody that targets CEA has been licensed in the treatment of colorectal cancer in humans by the clinical licensing authorities in the United Kingdom or United States. This, of course, includes MN-14.We acknowledge the in vitro and preclinical work that has been published using MN-14 (labetuzumab), which has been followed with interest over the years. A reference is actually made to this antibody in the introduction of our paper (Liersch et al, 2007; Conaghan et al, 2008). On a broader note, there are in fact over 200 antibodies that are under clinical testing in oncology (Reichert and Valge-Archer, 2007). Eight of these use CEA as a target, including T84.66, which, like MN14, have been used in clinical trials as radioconjugates (Wong et al, 2004; Reichert and Valge-Archer, 2007). Our paper certainly did not try and create an impression that PR1A3 was the only antibody to target CEA.It is interesting that 8 out of the 12 antibodies that are currently licensed for therapy in oncology are unconjugated, and that there are no conjugated antibodies licensed for therapy in solid tumours (Carter, 2006; Reichert and Valge-Archer, 2007). This may be a result of the poor outcomes in clinical trials of radioconjugates of murine antibodies in the 1980s. Conjugating antibodies to radioisotopes introduces the problem of bystander damage, complex technology involved in conjugation and the issue of adequate radiation delivery into solid tumours (Goldenberg, 2002; Sharkey and Goldenberg, 2005; Reichert and Valge-Archer, 2007).MN-14 has been used in preclinical studies. However, xenografts have the inherent problem of being a poor comparative model for antibody efficacy in humans (Wilkinson et al, 2001). This is based on two factors, abnormal vascularity of xenografts as well as the immunodeficient nature of the animals. Furthermore, there are differences in the murine and human immune system, which further complicates the matter, thereby raising major concerns about drawing parallels with what happens in humans. It is interesting that in the xenograft model, MN-14 was only effective in the GM-CSF treated group (Blumenthal et al, 2005). This cytokine is known to stimulate monocytes and promote their differentiation into macrophages. In mice, this cell type expresses FcγIV, which is homologous to FcγIII in humans (Nimmerjahn et al, 2005). A better in vivo model, which may better reflect antibody targeting, is a spontaneous tumour model in which immunocompetent MIN mice develop CEA-positive tumours (Wilkinson et al, 2001). We are currently in an advanced stage of testing unconjugated murine PR1A3 in this model.We feel that Blumenthal et al have failed to understand the main message of our paper, which relates to the importance of immune-mediated antibody responses. The emergence of immune-based mechanisms has become increasingly appreciated (Carter, 2006; Clynes, 2006). The results in our paper show that humanised IgG1 PR1A3 is able to elicit antibody-dependent cellular cytotoxicity (ADCC) against a range of human colorectal cancer cell lines using human effector cells (Conaghan et al, 2008). This is in agreement with the previous findings that MN-14 is able to trigger ADCC of CEA-positive colorectal cell lines, LoVo and LS174T (Blumenthal et al, 2005). Our study further defines NK cells as an important effector cell type in eliciting this response in humans. Significantly, PR1A3-induced NK-cell-mediated killing of colorectal cancer cells is not inhibited by free CEA, which is an important characteristic for any anti-CEA antibody to be successful in vivo. This can be explained by the specific binding of PR1A3 to membrane-bound CEA. Previous work has identified the B3-GPI anchor of CEA as being the epitope of PR1A3 (Durbin et al, 1994; Stewart et al, 1999). The authors feel that this information can be used to further engineer PR1A3 for maximal clinical effectiveness in humans. Like Blumenthal et al, we would envisage this happening in partnership with current chemotherapeutic regimens.\n\nREFERENCES:\n1. Blumenthal RD, Osorio L, Hayes MK, Horak ID, Hansen HJ, Goldenberg DM (2005) Carcinoembryonic antigen antibody inhibits lung metastasis and augments chemotherapy in a human colonic carcinoma xenograft. Cancer Immunol Immunother\n54: 315–32715592930\n2. Carter PJ (2006) Potent antibody therapeutics by design. Nat Rev Immunol\n6: 343–35716622479\n3. Clynes R (2006) Antitumor antibodies in the treatment of cancer: Fc receptors link opsonic antibody with cellular immunity. Hematol Oncol Clin North Am\n20: 585–61216762726\n4. Conaghan PJ, Ashraf SQ, Tytherleigh MG, Wilding JL, Tchilian E, Bicknell D, Mortensen NJ, Bodmer WF (2008) Targeted killing of colorectal cancer cell lines by a humanised IgG1 monoclonal antibody that binds to membrane-bound carcinoembryonic antigen. Br J Cancer\n98: 1217–122518349843\n5. Durbin H, Young S, Stewart LM, Wrba F, Rowan AJ, Snary D, Bodmer WF (1994) An epitope on carcinoembryonic antigen defined by the clinically relevant antibody PR1A3. Proc Natl Acad Sci USA\n91: 4313–43177514303\n6. Goldenberg DM (2002) Targeted therapy of cancer with radiolabeled antibodies. J Nucl Med\n43: 693–71311994535\n7. Granowska M, Jass JR, Britton KE, Northover JM (1989) A prospective study of the use of 111In-labelled monoclonal antibody against carcino-embryonic antigen in colorectal cancer and of some biological factors affecting its uptake. Int J Colorectal Dis\n4: 97–1082746136\n8. Hajjar G, Sharkey RM, Burton J, Zhang CH, Yeldell D, Matthies A, Alavi A, Losman MJ, Brenner A, Goldenberg DM (2002) Phase I radioimmunotherapy trial with iodine-131-labeled humanized MN-14 anti-carcinoembryonic antigen monoclonal antibody in patients with metastatic gastrointestinal and colorectal cancer. Clin Colorectal Cancer\n2: 31–4212453334\n9. Liersch T, Meller J, Bittrich M, Kulle B, Becker H, Goldenberg DM (2007) Update of carcinoembryonic antigen radioimmunotherapy with (131)I-labetuzumab after salvage resection of colorectal liver metastases: comparison of outcome to a contemporaneous control group. Ann Surg Oncol\n14: 2577–259017570017\n10. Liersch T, Meller J, Kulle B, Behr TM, Markus P, Langer C, Ghadimi BM, Wegener WA, Kovacs J, Horak ID, Becker H, Goldenberg DM (2005) Phase II trial of carcinoembryonic antigen radioimmunotherapy with 131I-labetuzumab after salvage resection of colorectal metastases in the liver: five-year safety and efficacy results. J Clin Oncol\n23: 6763–677016170184\n11. Nimmerjahn F, Bruhns P, Horiuchi K, Ravetch JV (2005) FcgammaRIV: a novel FcR with distinct IgG subclass specificity. Immunity\n23: 41–5116039578\n12. Reichert JM, Valge-Archer VE (2007) Development trends for monoclonal antibody cancer therapeutics. Nat Rev Drug Discov\n6: 349–35617431406\n13. Sharkey RM, Goldenberg DM (2005) Perspectives on cancer therapy with radiolabeled monoclonal antibodies. J Nucl Med\n46(Suppl 1): 115S–127S15653660\n14. Sharkey RM, Juweid M, Shevitz J, Behr T, Dunn R, Swayne LC, Wong GY, Blumenthal RD, Griffiths GL, Siegel JA, Leung S, Hansen HJ, Goldenberg DM (1995) Evaluation of a complementarity-determining region-grafted (humanized) anti-carcinoembryonic antigen monoclonal antibody in preclinical and clinical studies. Cancer Res\n55: 5935s–5945s7493374\n15. Stewart LM, Young S, Watson G, Mather SJ, Bates PA, Band HA, Wilkinson RW, Ross EL, Snary D (1999) Humanisation and characterisation of PR1A3, a monoclonal antibody specific for cell-bound carcinoembryonic antigen. Cancer Immunol Immunother\n47: 299–30610203059\n16. Wilkinson RW, Ross EL, Poulsom R, Ilyas M, Straub J, Snary D, Bodmer WF, Mather SJ (2001) Antibody targeting studies in a transgenic murine model of spontaneous colorectal tumors. Proc Natl Acad Sci USA\n98: 10256–1026011517330\n17. Wong JY, Chu DZ, Williams LE, Yamauchi DM, Ikle DN, Kwok CS, Liu A, Wilczynski S, Colcher D, Yazaki PJ, Shively JE, Wu AM, Raubitschek AA (2004) Pilot trial evaluating an 123I-labeled 80-kilodalton engineered anticarcinoembryonic antigen antibody fragment (cT84.66 minibody) in patients with colorectal cancer. Clin Cancer Res\n10: 5014–502115297402"
+}

batch_11/PMC2528308.json

@@ -0,0 +1,4 @@
+{
+    "id": "PMC2528308",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2528308\nAUTHORS: B. Miedema, J. Easley, P. Fortin, R. Hamilton, M. Mathews\n\nABSTRACT:\nObjectiveIn a study conducted in New Brunswick and Newfoundland and Labrador, we examined the economic impact on families caring for a child with cancer.MethodsWe undertook semi-structured interviews with 28 French and English families with a child diagnosed with cancer in the last 10 years.ResultsFamilies who care for a child with cancer incur considerable costs during the diagnostic, treatment, and follow-up care phases of the disease. Four major themes emerged from this qualitative study as contributing factors for these expenses: necessary travel; loss of income because of a reduction or termination of parental employment; out-of-pocket treatment expenses; and inability to draw on assistance programs to supplement or replace lost income. In addition, many of the decisions with regard to the primary caregiver were gendered. Typically, the mother is the one who terminated or reduced work hours, which affected the entire family’s financial well-being.ConclusionsFor families with children diagnosed with cancer, financial issues emerged as a significant concern at a time when these families were already consumed with other challenges. This economic burden can have long-term effects on the financial security, quality of life, and future well-being of the entire family, including the siblings of the affected child, but in particular the mother. Financial assistance programs for families of seriously ill children need to be revisited and expanded.\n\nBODY:\n1. INTRODUCTIONIn Atlantic Canada, approximately 90 children between birth and 14 years of age are diagnosed with cancer annually 1. Although childhood cancer is rare, it is nevertheless the most common disease-related cause of death among children. Fortunately, the survival rate for children with cancer has increased dramatically since the late 1990s. Understandably, the psychological, sociologic, and financial effects of the disease can be extremely stressful for families 2. Few studies have been conducted to document these issues, particularly from the perspective of the families who care for a child with cancer.2. BACKGROUNDThe experience of pediatric cancer patients is different from that of adults with cancer, because the whole family—particularly the parents, and in some cases, the grandparents—are usually completely involved in the child’s illness 3,4. It has been reported that parents can develop posttraumatic stress disorder from dealing with a child’s illness 5,6. Siblings have reported feeling lost and ignored by parents who are preoccupied with the sick child and who may be absent from home for extended periods of time accompanying a child receiving treatment out of town. These feelings can lead to behavioural challenges in the siblings left at home 3.In addition to the disruptions of family dynamics, families describe financial hardship associated with caring for a child with cancer. In one study, 37% of families reported that they were forced to borrow money to cover the extra cost of treatment related to the child’s illness 7. Another study reported that parents of children with cancer suffered greater financial hardship than did parents of children with other serious illness such as diabetes 8.Barr and Sala 9 reported that few studies have specifically examined the out-of-pocket expenses incurred by families dealing with childhood cancer and other chronic diseases. A small qualitative Canadian study by Scott–Findlay and Chalmers 10 reported that, among other hardships, families with children who had cancer were required to travel 400 km on average (round trip) to receive treatment. Yantzi et al. 11 reported a relationship between the distance a family has to travel to the hospital for children with chronic illness and the quality of family relationships, because of the travel time and time spent away from home.In the present study, we were interested in learning about the experiences of families in New Brunswick and Newfoundland and Labrador who had cared or were caring for a child with cancer, and the effect of this experience on the family’s financial well-being.2.1 Study SettingNew Brunswick and Newfoundland and Labrador are unique in that, in both provinces, half the population lives in rural areas or small towns. As a result, many people must travel to receive specialized treatments. For example, in New Brunswick, pediatric cancer patients are usually treated out-of-province in either Nova Scotia or Quebec. In Newfoundland and Labrador, patients can attend the Janeway Hospital for Children in St. John’s, but the province is large, and travel from remote areas can involve journeys of hundreds of kilometres.2.2 Health Care SystemAll Canadian residents are entitled to enrol in a provincial health plan (php). The php covers all medically necessary physician and hospital costs and the cost of drugs provided in hospital.Prescription drugs provided outside a hospital setting are not covered by the phps. For cancer patients, these drugs may include oral chemotherapy agents that can be administered at home 12 or supportive drugs (such as antiemetics or pain medications) given to combat the side effects of treatment. Provincial drug insurance programs may offset the costs of some of these drugs for low-income families, and both New Brunswick and Newfoundland and Labrador have such programs 13. Out-of-pocket drug expenses may also be cost-shared through private supplemental health insurance programs (Blue Cross or Medavie, for example). Private supplemental medical insurance (including a prescription drug plan) is often offered as an employment benefit, and individuals can also purchase supplemental health insurance on their own (but usually at much higher premiums). Costs covered through private supplemental insurance policies vary and may require a 20%–30% co-payment 14. Overall, 20% of Canadians lack private supplemental health insurance, and the proportion is higher in the Atlantic Provinces 15. In 2001, it was reported that 30% of Newfoundland and Labrador residents and 32% of New Brunswick residents did not have private supplemental health insurance 16.3. METHODSA qualitative research method was chosen for this project. Using qualitative methods, researchers can study social and cultural phenomena in the context of people’s day-to-day lives and from the viewpoint of the participants. This approach permits researchers to address the uniqueness of the particular situation and to generate a hypothesis or theory 17.3.1 Research Ethics Board CertificationThe research protocol used in our study was reviewed and approved by the River Valley Health Research Ethics Committee and the research ethics boards of Memorial University of Newfoundland and Université de Moncton. All participants signed an informed consent form before commencing their interview.3.2 Inclusion CriteriaFor inclusion in the study, we recruited parents or caregivers whose children were 19 years or younger when diagnosed with cancer. The child’s diagnosis had to have occurred no more than 10 years before recruitment.3.3 Recruitment ProcessIn New Brunswick, staff members at the Canadian Cancer Society–New Brunswick Division mailed a letter to the parents of children who had participated in summer camps for children with cancer. The parents, if interested, could directly contact the study team for more information and to set up an interview. Participants were also recruited through newspaper articles and other French and English media.In Newfoundland and Labrador, participants were recruited with the assistance of Candlelighters Canada–Newfoundland and Labrador Division, a childhood cancer support foundation. A notice of invitation was posted in the Candlelighters newsletter, followed by a letter sent to 25 specific families that fit the inclusion criteria.The recruitment phase was concluded when the research team agreed that demographic and linguistic diversity was achieved in the sample and that no new themes were emerging from the interviews. With a total response rate to the individual mailings of approximately 30%, 9 anglophone and 12 francophone parents in New Brunswick and 7 anglophone parents in Newfoundland and Labrador participated in the study. Participants included one or both parents or caregivers.3.4 The InterviewA semi-structured format was used to guide the interviews with the participants. The interview questions were all open-ended. The interview schedule started with general questions and then moved to questions about social supports, the effect of cancer on the child and the family, and specific questions about the economic effects of cancer. Most interviews took place in the participants’ homes, although a few took place in one of the research offices. In general, the interviews lasted between 1 and 2 hours. All participants agreed to have their interviews audiotaped on a digital recorder. After the interview was completed, the participants were asked to complete a sociodemographic form.3.5 Data AnalysisAll interviews were transcribed verbatim, and a rigorous constant comparative thematic analysis was applied 18. In a thematic analysis, researchers identify themes and common patterns among the experiences of the participants 19,20.The three researchers and their three assistants all read 6 selected transcripts representing the three distinct interview groups: English New Brunswick, French New Brunswick (translated transcripts), and English Newfoundland and Labrador. All six researchers coded the transcripts independently. The researchers then convened for a 2-day team meeting to discuss the codes. Most codes were easily agreed on; in cases where disagreement arose, team discussions ensued to reach a consensus about the code. An English coding scheme was developed, and this coding scheme was used to analyze the rest of the transcripts from all three interview groups. The coding scheme was slightly revised and updated during two subsequent teleconferences.Of the French interviews, 8 were translated into English by an official translation agency. Four interviews were coded in French by a bilingual researcher using the English coding scheme.3.6 ConfidentialityBecause of the relatively small number of pediatric cancer cases and the small population in the two provinces, as little detail as possible regarding the identities of the study participants is revealed here. Quotes may have been slightly modified to ensure that no identifying information is disclosed.4. RESULTS4.1 Profile of the Study PopulationOf the 28 families, 5 had children still in active treatment at the time of the interview, 3 had experienced the death of their child, and the rest had children in the follow-up care stage of the cancer care continuum. Just over half (57%) of the families lived in rural areas, and almost three quarters considered themselves to be religious (68%) at the time of the interview. Most of the parents were married, with an average of 2 children, and they had postsecondary educations and good incomes (see Table I).Among the parents, 17 mothers (61%) were working full- or part-time and 11 (39%) were not working (1 retired, 7 not in the labour force, 3 on sick leave or stress leave) at the time of the interview; 24 fathers (86%) were working full- or part-time, 2 (7%) were seasonal workers, and 2 (7%) were not in the workforce.The age of the affected children at the time of diagnosis in New Brunswick ranged from 6 months to 17 years. In Newfoundland and Labrador, the age of the affected children at diagnosis ranged from 3 to 16 years. The most common diagnosis was acute lymphocytic leukemia. All children had received chemotherapy, a few had received radiation therapy, and a few had undergone bone marrow transplantation.4.2 Themes Related to Economic EffectsThe four major themes that emerged as contributing factors to the severe economic effects on the families weretravel expenses for treatment and follow-up care,loss of income because of a reduction or termination of parental employment,out-of-pocket expenses for treatment, andan inability to draw on programs for assistance or income supplementation.4.2.1 Travel CostsOf the 28 families interviewed, all but 3 were required to travel to other cities, frequently out-of-province, for treatments. In Newfoundland and Labrador, all families traveled to the Janeway Children’s Hospital in St. John’s. Most of the New Brunswick families traveled to the iwk Health Centre in Halifax, Nova Scotia. Some families living in western and northern New Brunswick traveled to children’s hospitals in Quebec. A few parents whose children underwent very specialized treatments such as bone marrow transplantation, traveled to Ontario hospitals.It was not uncommon for parents to report having to travel immediately after a medical consultation to a large children’s hospital without returning home for weeks or sometimes months. Parents were then forced to make alternative arrangements for other children at home and (if they were working for pay) with their employers. As one parent said, “We just had to jump in the van and drive to [city] with literally the clothes on our backs and a few dollars. It was all we had. When we got there, we did not think about where we [were] going to stay” (interview 7).The costs associated with travel and accommodations were substantial for many parents. One father said, “Financially, it set us back 10 years because of the loss of my salary and the wretched trips” (interview 9). Many families were able to take advantage of reduced-rate accommodations such as the Ronald McDonald House or hospital rates at local hotels; however, because of prolonged stays, the costs still added up for these families. As one mother described it, “even though you had Ronald MacDonald house to stay at for $11 a night, of course the phone bill[....] Every day, every report, we phoned home. Parking, meals at the hospital, and we tried to get groceries and eat when we could at Ronald MacDonald house, but that was ... you know, if I was going to take a guess, I would say it was couple thousand dollars for those three weeks” (interview 14).4.2.2 Work-Related IssuesIn this study, caring for a child with cancer greatly affected the work patterns of the parents in general, but particularly the work patterns of the mothers. The work status of the parents at the time of their child’s cancer diagnosis and during treatment shows a considerable gendered change (see Figures 1 and 2). At the time of diagnosis, 24 fathers and 22 mothers were working for pay either full-time or part-time. Of the fathers, 61% reported that their work hours changed during the child’s treatment; among the mothers, the proportion was 86%.  Five mothers (18%) simply stopped working altogether.One father described why his wife stopped working and looked after their sick child: “[She] is a nurse, and our salaries are pretty much equal. She earns a little bit more than I do[....] If I left for a year, six months or two years, I would have to transfer a lot of the projects to somebody else. It would be hard for me to start again, but it seems to be a lot easier for her [...] plus, she’s the mother.” During treatment, this mother was unable to continue to work. The father said, “[She] tried to work through [the child’s treatment] for a few months, and then she had to go on sick leave[....] The company denied her benefits. They said, ‘You’re not sick, your son is.’” (interview 13).Self-employed parents often experienced an immediate loss of income: “There were times, you know, when we weren’t able to work the hours that we normally work, so there was much less money coming in[....] If you do not go to work, you don’t get paid” (interview 2).4.2.3 Out-of-Pocket Medical CostsNot only do travel, lodging, and meals away from home add to costs, but so too do medications and medical supplies not covered by the php. Private supplemental health insurances covers some medication and supply costs, but parents without insurance must pay for out-of-hospital drugs and medical supplies themselves. Many of the interviewees spoke of spending many thousands of dollars for treatment equipment such as feeding tubes, needles, and medication. One mother was so overwhelmed by these costs for medical supplies for her child that she felt “the last thread snapped” when she was negotiating with the supplemental private health insurer on what they would cover and what they would not cover. This mother was ultimately forced to cancel her daughter’s Registered Education Savings Plan and to withdraw the funds to pay for medical expenses.4.2.4 Inability to Draw On Income Support ProgramsFew parents were able to draw on formal programs for financial support. Only 1 parent remarked that supplemental private health insurance provided a per diem when the child was hospitalized; in most cases, however, the parents were unable to find any respite from the demands on their finances. Many parents reported that programs were contingent on the parent being able to “look for work” and that financial assistance was based on “previous earnings.”One mother described her struggle with the federal Employment Insurance program: “We [husband and wife] tried to get on unemployment, but we couldn’t get that. We tried to get social assistance to help us, and they did a little bit, but it wouldn’t be enough to butter your bread, because they had said something about ‘You made too much money the month before.’” She noted that her husband had been laid off just before their daughter’s diagnosis of cancer: “They gave us the weekend off to pack up and to drive to the iwk. We went to the unemployment office to see if they would help us because he was laid off, but they refused. We did not lie about anything. I told him that my daughter had just found out that she has cancer, and we are leaving for Halifax. But they said there is nothing we can do[....] So you go with the credit cards you have” (interview 3).5. DISCUSSION AND CONCLUSIONSOur research clearly demonstrates the severity of the negative financial effects on families in New Brunswick and Newfoundland and Labrador when they care for a child with cancer. The emerging theory from this research is that government programs are inadequate to support families who care for children with catastrophic illnesses such as cancer. Although none of the interviewed families suggested that they had to withdraw care from their child because of financial constraints, we demonstrated that many parents struggled with financial hardship and that these concerns imposed additional stresses on the families.Debts accrued over the course of the treatment, and the follow-up phase of the disease could have long-term effects on the financial stability of the family. Many families discussed how they were still paying off debts years after the treatment. A few parents even discussed having to re-mortgage their homes or to take money out of registered retirement or education savings plans to pay for the medical and out-of-pocket expenses.Care for a child with cancer was, not surprisingly, gendered. It was most often the mother who reduced or terminated employment to care for the sick child, regardless of prior earning power in the family. Her reduced income not only was responsible for the immediate drop in family income, but also potentially jeopardized her future and retirement earnings. Inability to contribute to a registered retirement savings plan or a company pension plan could also affect her income in old age 21.Caregivers in the formal health care system need to be aware that parents of pediatric cancer patients not only have to deal with the stress of the illness, but also with stressors in their immediate environment, which can be very severe 6,11.Canada lacks support programs for parents caring for a child with a catastrophic illness. The federal government has introduced a “compassionate leave” program, but that program is geared toward caring for a spouse or parent with a terminal illness 22. Parents who care for a child with cancer are particularly vulnerable to financial ruin at the time of cancer treatments and, in most cases, for many years afterward, because cancer survivorship for children increases with technological and medical advances. Canada needs to develop programs for parents who care for children with catastrophic illnesses so that parents do not have to struggle financially to properly care for the child with cancer and that child’s siblings.FIGURE 1Employment of parents of children with cancer at the time of diagnosis.FIGURE 2Employment status change of parents of children with cancer during the treatment phase.TABLE IIncome and education of parents of children with cancer at time of interviewNBNLTotal [n (%)]Education Mother  High school or less224 (14)  Professional diploma437 (25)  Undergraduate degree617 (25)  Graduate/professional degree819 (32)  Did not wish to answer101 (04) Father  High school or less639 (32)  Professional diploma336 (21)  Undergraduate degree617 (25)  Graduate/professional degree505 (18)  Did not wish to answer101 (04) Annual family income  $20,000 or less011 (04)  $20,001 to $30,000404 (15)  $30,001 to $40,000000  $40,001 to $50,000314 (15)  $50,001 to $60,000213 (11)  $60,001 to $70,000112 (08)  $70,001 to $80,000202 (08)  $80,001 or more729 (32)  Did not wish to answer213 (11)NB = New Brunswick; NL = Newfoundland and Labrador.\n\nREFERENCES:\nNo References"
+}

batch_11/PMC2528557.json

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+{
+    "id": "PMC2528557",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2528557\nAUTHORS: M.D. Duncan, A. Leis, J.W. Taylor–Brown\n\nABSTRACT:\nBackgroundIndividuals have increasingly sought complementary therapies to enhance health and well-being during cancer, although little evidence of their effect is available.ObjectivesWe investigated\n how an Iyengar yoga program affects the self-identified worst symptom in a group of participants. whether quality of life, spiritual well-being, and mood disturbance change over the Iyengar yoga program and at 6 weeks after the program. how, from a participant’s perspective, the Iyengar yoga program complements conventional cancer treatment.Patients and MethodsThis pre–post instrumental collective case study used a mixed methods design and was conducted at a private Iyengar yoga studio. The sample consisted of 24 volunteers (23 women, 1 man; 88% Caucasian; mean age: 49 years) who were currently on treatment or who had been treated for cancer within the previous 6 months, and who participated in ten 90-minute weekly Iyengar yoga classes.The main outcome measures were most-bothersome symptom (Measure Your Medical Outcome Profile 2 instrument), quality of life and spiritual well-being (Functional Assessment of Chronic Illness Therapy–General subscale and Spiritual subscale), and mood disturbance (Profile of Mood States–Short Form). Participant perspectives were obtained in qualitative interviews.ResultsStatistically significant improvements were reported in most-bothersome symptom (t(23) = 5.242; p < 0.001), quality of life (F(2,46) = 14.5; p < 0.001), spiritual well-being (F(2,46) = 14.4; p < 0.001), and mood disturbance (F(2,46) = 10.8; p < 0.001) during the program. At follow-up, quality of life (t(21) = −3.7; p = 0.001) and mood disturbance (t(21) = 2.4; p = 0.025) significantly improved over time. Categorical aggregation of the interview data showed that participants felt the program provided them with various benefits not included on the outcomes questionnaires.ConclusionsOver the course of the Iyengar Yoga for Cancer program, participants reported an improvement in overall well-being. The program was also found to present participants with a holistic approach to care and to provide tools to effectively manage the demands of living with cancer and its treatment.\n\nBODY:\nNo Body Content\n\nREFERENCES:\nNo References"
+}

batch_11/PMC2528562.json

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+{
+    "id": "PMC2528562",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2528562\nAUTHORS: S.M. Sagar, A.M. Leis\n\nABSTRACT:\nNo Abstract\n\nBODY:\nWe are pleased to present this special supplement of Current Oncology, which is generously financed by a grant from the Lotte and John Hecht Memorial Foundation. Integrative oncology is both a science and a philosophy that focuses on the complexity of the health of cancer patients and proposes a multitude of approaches to accompany the conventional therapies of surgery, chemotherapy, molecular therapeutics, and radiotherapy to facilitate health. As such, integrative oncology involves thinking outside the box, and so we are indeed fortunate to have attracted a plethora of manuscripts from innovative leaders both of Canadian and of international cancer treatment and control services.In line with the modern approach to media communications, we have “integrated” this issue with manuscripts published on the Current Oncology Web site. Readers will also have the opportunity to view and listen to slide presentations submitted to the Integrating Wellness into Cancer Care Conference held at the University of Toronto, October 4–5, 2007. The conference was organized by Dr. Paul Fortin in memory of his wife Dr. Veronique Benk. Veronique was a radiation oncologist, clinician, and researcher who specialized in breast cancer, and she was devoted to her patients. Her personal experience of breast cancer and myeloid leukemia was transformative, and she embraced a wider approach to cancer treatment. That approach prioritized state-of-the-art medical care with a new emphasis on spirituality, wellness, and quality of life. The conference was sponsored by a non-restricted educational grant from the Lotte and John Hecht Memorial Foundation, CV Technologies, Astra/Zeneca Pharmaceuticals, Pfizer Pharmaceuticals, Novartis Pharmaceuticals, Wellspring, and the University of Toronto Department of Radiation Oncology, with special thanks to Drs. Mary Gospodarowicz and Pamela Catton.PURSUING THE INTEGRATIVE PATHSo, what is cam?Complementary and alternative medicine (cam) is an umbrella term encompassing a group of diverse medical and health care systems, practices, and products that are not always considered part of conventional medicine 1. Yet in traditional healing systems, the power of the mind and other non-pharmaceutical interventions are integral to the treatment of ill health.Promotion, maintenance, monitoring, or restoration of health is the goal of cam use. According to the U.S. National Cancer Institute, cam includes whole medical systems (for example, Traditional Chinese Medicine), mind–body medicine (meditation, for instance), biologically-based practices (natural health products, among others), manipulative and body-based therapies (for example, massage), and energy therapies (qi gong, for instance) 1.An approach is called “complementary” when it is used adjunctively to conventional treatments, with the intent to enhance the body’s natural abilities to heal. Modalities used instead of conventional treatments are labelled “alternative” therapies. The term “alternative” means that treatments outside of conventional medicine are used to treat the disease. Depending on intent, some therapies can be considered either complementary or alternative. It is therefore important always to clarify the intent of the patient considering cam use. Studies have consistently found that most cancer patients use cam as a complement to their conventional treatment 2,3.The lack of consensus around terminology and definitions makes it difficult to accurately assess the prevalence of cam use. For example, the list of products and therapies designated as cam continually changes as therapies that are proved to be safe and effective are integrated into conventional health care and as new, untested ones emerge 4.An increasing number of people living with cancer are using therapies in addition to those prescribed by conventional health care providers 5,6. In the literature, an explosion of cam surveys has suggested that utilization rates by cancer patients fall between 40% and 60% 7. For example, with the inclusion of prayer in the cam list, the prevalence of cam use in the United States is estimated at 62%; minus prayer, it is about 36% 8.Integrative oncology does not usually incorporate prayer into its definition of therapies, preferring to classify prayer in a religious domain, but the concept of prayer as a mind–body intervention illustrates the challenge of defining a cam therapy 9. Throughout the cancer trajectory, higher use of cam is consistently found during chemotherapy to mitigate adverse effects, after conventional cancer treatment to boost energy, during survivorship to foster wellness, and during the last months of life to control symptoms 10.For this special issue, the term “cam” is being used because of its worldwide recognizability as a label linked to traditional medicine. Given the specific focus of this issue on integrative medicine, cam uniquely refers to the complementary side—in other words, to something used in conjunction with conventional oncology treatments. The powerful synergy of the holistic approach of complementary medicine together with biomedical cancer treatments is central to the purpose of integrative oncology. The combination permits dysfunctional physical, mental, emotional, and spiritual symptoms to be treated and thereby fully addresses the healing needs of cancer patients in a tailored fashion.IN THIS SUPPLEMENTIn his article, Dr. Simon Sutcliffe discusses the importance of combining science and evidence-based medicine with individual and societal values and of integrating values into a process of holistic care. To quote Sutcliffe, the goal is ultimately “to achieve a responsive, efficient, effective, and sustainable system to improve health and control cancer (as a process, not as an event).” He expands his argument that complex problems require a more sophisticated approach than can be achieved through scientific reductionism, and that the multifaceted perspectives of the patient must be part of the decision-making process in designing health policy.Drs. Jacqueline Bender and Alejandro Jadad extend the perspective of patients’ values and education through individuals socializing over the Internet. They discuss the potential for empowerment and how that empowerment may modify the relationships of patients with their health care providers. Dr. Alastair Cunningham emphasizes the existential crisis that patients endure when they receive a diagnosis of cancer and speaks of the importance of psychological healing as part of the process of restoring health. And Dr. Mary Vachon expands on the notion of spirituality and meaning for cancer patients in her article “The Soul’s Wisdom: Stories of Living and Dying” (e-manuscript on the Web).How is outcome to be evaluated in an integrative oncology program? Dr. Stephen Sagar discusses various health outcome domains and patient satisfaction, and points to some validated measurement tools. For a detailed source of measurement tools, readers are also directed to the new online IN-CAM Outcomes Database (www.outcomesdatabase.org) organized by Dr. Marja Verhoef.Dr. Sagar and Dr. Raimond Wong together provide an educational article on integrative oncology research and regulation (e-manuscript on the Web). Further e-manuscripts recount the experiences of two international integrative oncology programs: Dr. Jane Maher, Chief Medical Officer of MacMillan Cancer Support, describes the Lynda Jackson Macmillan Centre at Mount Vernon Hospital in Northwood, United Kingdom, and Dr. Gary Deng describes the Integrative Medicine Service at Memorial Sloan–Kettering Hospital, headed by Dr. Barrie Cassileth, in New York City, United States. Both centres have pioneered similar models in other countries.Attendees of the University of Toronto Integrating Wellness into Cancer Care Conference participated in a workshop titled “How to Put Wellness on the Prescription Pad”, and in another e-manuscript, Dr. Fortin presents a summary of the discussions that took place.A group of manuscripts from the Cancer, Complementary and Alternative Medicine (ccam) team, a multidisciplinary group of Canadian scientists, presents some of their work in evaluating the role of complementary therapies for cancer care.Dr. Ann Leis discusses the scope of integrative oncology and the need for evidence to support the integration of complementary therapies into cancer care. She concludes that “a whole-systems framework to the development of the evidence base for integrative oncology can guide the development of evidence that respects the complex nature of many complementary and integrative practices and their underlying principles of care delivery.”In her manuscript “Talking to Cancer Patients About Complementary Therapies,” Marja Verhoef concludes that discussing cam with patients is the physician’s responsibility and that it will facilitate evidence-based, patient-centred cancer care.Dr. Lynda Balneaves uses research from the Canadian health literature to address the issue of patient decision-making. In her discussion, she says that “decision to use, or not to use, cam is not a one-time whimsical decision; instead, it is a decision that leads cancer patients to reflect on their unique personal and social context and to ponder how cam may fit with their values, beliefs, and specific health care needs. As the individual and social contexts of patients change, the appropriateness of select cam therapies in their treatment regimen also changes. Decisions about cam are not static; rather, they are dynamic entities that require assessment and follow-up by health professionals throughout a patient’s illness.”Why do some patients decline conventional evidence-based therapies and pursue alternative non-proven options? Dr. Verhoef concludes that “poor doctor–patient communication, the emotional impact of the cancer diagnosis, perceived severity of conventional treatment side effects, a high need for decision-making control, and strong beliefs in holistic healing appear to affect the decision by patients to decline some or all conventional cancer treatments.” The “tendency by doctors to dichotomize patient decisions as rational or irrational may interfere with the ability of the doctors to respond with sensitivity and understanding,” she continues.Doctor of naturopathic medicine Dugald Seeley, together with Dr. Doreen Oneschuk, discusses the important topic of interactions of natural health products with biomedical cancer treatments.These subjects require knowledgeable physicians and a modification of medical school curricula, a task that is being undertaken by the CAM in UME Project (www.caminume.ca/about.html) and the Consortium of Academic Health Centers for Integrative Medicine (www.imconsortium.org).What, then, are the current integrative practices of Canadian health care professionals? Dr. Alison Brazier uses an interpretive–description research design, with a series of in-depth qualitative interviews, to highlight two main strategies: acting as an integrative cancer guide and collaborating with other health care professionals.Mind–body techniques derived from Eastern mysticism have become an important part of some integrative oncology programs. In a research paper that evaluates an Iyengar yoga program, Ms. Meghan Duncan finds an overall improvement in the well-being of cancer patients (e-manuscript on the Web). This research contributes further to the evidence that some techniques derived from Eastern spirituality can help some patients cope with cancer and its treatment.THE WAY FORWARDThe future of complementary therapies lies in mainstream medicine, but only if those therapies are based on scientific understanding and evidence of effectiveness. A willingness to discard therapies that fail to be proved effective in clinical studies is vital. Accepted therapies must also be seen to be safe and cost-effective.A comprehensive cancer program should integrate surgery, chemotherapy, radiotherapy, and targeted molecular therapies with meaningful psycho-spiritual, psychological, and physical supportive therapies, and an investigative program of botanicals. New technology is facilitating the quality-controlled preparation of simple and complex mixtures of phytochemicals that are being investigated as biologic response modifiers. International collaboration between North American, European, and Asian universities and interested pharmaceutical companies is encouraging the development of multi-targeted therapies using traditional herbs. During a recent visit by S.M.S. to Fudan University in Shanghai, China, that institution ratified a collaborative research agreement with the M.D. Anderson Hospital (the largest international cancer centre) and signed a new collaborative agreement with the Institut Gustav Roussy (Europe’s largest cancer centre), under the umbrella of integrative oncology. Video extracts of the Shanghai conference of the Society for Integrative Oncology can be found on the Current Oncology Web site. Further information on the Society for Integrative Oncology and its conferences (including abstracts from its recent conference held in Shanghai) can be found at www.integrativeonc.org.YOUR PART IN THE DISCUSSIONReaders are invited to evaluate this special supplement by answering a short survey on the Current Oncology Web site. The editors appreciate your interest in this supplement and will similarly appreciate receiving your comments. We would like to publish those comments on the Web site; the option of anonymity for specific comments is available.\n\nREFERENCES:\n1. National Institutes of Health, National Center for Complementary and Alternative Medicine (nccam)Health information > cam basics > What is cam? [Web page]Gaithersburg, MDNCCAMFebruary 2008[Available at: nccam.nih.gov/health/whatiscam; cited: March 27, 2008]\n2. RichardsonMASandersTPalmerJLGreisingerASingletarySEComplementary/alternative medicine use in a comprehensive cancer center and the implications for oncologyJ Clin Oncol20001825051410893280\n3. LewithGWalachHJonasWBLewithGWalachHJonasWBBalanced research strategies for complementary and alternative medicineClinical Research in Complementary Therapies: Principles, Problems and SolutionsEdinburghChurchill Livingstone2002\n4. ErnstEPittlerMHWiderBBoddyKComplementary/ alternative medicine for supportive cancer care: development of the evidence baseSupport Care Cancer200715565817093910\n5. PaltielOAvitzourMPeretzTDeterminants of the use of complementary therapies by patients with cancerJ Clin Oncol20011924394811331323\n6. KolstadARisbergTBremnesYUse of complementary and alternative therapies: a national multicentre study of oncology health professionals in NorwaySupport Care Cancer2004123121814767750\n7. MolassiotisAFernadez–OrtegaPPudDUse of complementary and alternative medicine in cancer patients: a European surveyAnn Oncol2005166556315699021\n8. BarnesPMPowell-GrinerEMcFannKNahinRLComplementary and alternative medicine use among adults: United States, 2002Adv Data20042711915188733\n9. SloanRPBagiellaEPowellTReligion, spirituality, and medicineLancet1999353664710030348\n10. ShenJAndersenRAlbertPSUse of complementary/alternative therapies by women with advanced-stage breast cancerBMC Complement Altern Med20022812175424"
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batch_11/PMC2528934.json

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+{
+    "id": "PMC2528934",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2528934\nAUTHORS: Joseph Donroe, Monica Tincopa, Robert H. Gilman, Doug Brugge, David A. J. Moore\n\nABSTRACT:\nBackgroundChild pedestrian road traffic injuries (RTIs) are an important cause of death and disability in poorer nations, however RTI prevention strategies in those countries largely draw upon studies conducted in wealthier countries. This research investigated personal and environmental risk factors for child pedestrian RTIs relevant to an urban, developing world setting.MethodsThis is a case control study of personal and environmental risk factors for child pedestrian RTIs in San Juan de Miraflores, Lima, Perú. The analysis of personal risk factors included 100 cases of serious pedestrian RTIs and 200 age and gender matched controls. Demographic, socioeconomic, and injury data were collected. The environmental risk factor study evaluated vehicle and pedestrian movement and infrastructure at the sites in which 40 of the above case RTIs occurred and 80 control sites.FindingsAfter adjustment, factors associated with increased risk of child pedestrian RTIs included high vehicle volume (OR 7·88, 95%CI 1·97–31·52), absent lane demarcations (OR 6·59, 95% CI 1·65–26·26), high vehicle speed (OR 5·35, 95%CI 1·55–18·54), high street vendor density (OR 1·25, 95%CI 1·01–1·55), and more children living in the home (OR 1·25, 95%CI 1·00–1·56). Protective factors included more hours/day spent in school (OR 0·52, 95%CI 0·33–0·82) and years of family residence in the same home (OR 0·97, 95%CI 0·95–0·99).ConclusionReducing traffic volumes and speeds, limiting the number of street vendors on a given stretch of road, and improving lane demarcation should be evaluated as components of child pedestrian RTI interventions in poorer countries.\n\nBODY:\nIntroductionRoad traffic injuries (RTIs) are an important cause of morbidity and mortality, and are projected to become the sixth leading cause of death and third leading cause of disability adjusted life years (DALYs) lost globally by the year 2020 [1]. Poorer nations are disproportionately affected by RTIs and account for approximately 85% of RTI deaths and 90% of RTI disability [2]. In poorer countries of Latin America, RTIs are already the sixth leading cause of death and third leading cause of morbidity for all ages [3]. While well-designed research, successful interventions, and legislative priority has led to a substantial decrease in the burden of RTIs in wealthier regions, the rates of RTIs in many poorer nations are increasing [4].Children and pedestrians are especially vulnerable to traffic injuries, particularly in developing countries [5], [6], [7], [8], [9]. In the low to middle income countries of the Americas, RTIs are the number one cause of death and morbidity for children aged 5–14, and a leading cause of death for children aged 0–4 [3]. Additionally, the RTI fatality rate for children of poorer countries is as much as six times that of children from high income countries [5]. Pedestrians are involved most frequently in RTIs in the developing world, and represent up to 54% of those injured in Latin American studies [8], [10], [11].Prevention of child pedestrian RTIs has focused on modifying both personal (education initiatives) and, more effectively, environmental (traffic calming) risk factors [12], [13], [14], [15], [16], [17], [18]. Environmental risk factors themselves, however, have been less rigorously studied [19]. The personal risk factors encountered in the literature include age, gender, household overcrowding, poverty, single parent homes, and low levels of education in caregivers, while environmental risk factors include high traffic volumes, high vehicle speeds, presence of sidewalks, and density of curb side parking [12], [15], [16], [17], [19], [20], [21], [22], [23], [24], [25]. The overwhelming majority of these studies were conducted in developed countries [4], [26], and the results are commonly relied upon when importing or creating intervention strategies for the developing world. The assumption, however, that developed world practices translate into effective prevention measures in poorer countries may be erroneous as they may not be affordable, may require disproportionate technologies, and may miss important risk factors unique to developing world settings [14], [27], [28].The aim of this study was to assess personal and environmental risk factors for child pedestrian RTIs in the urban, developing world setting of Lima, Peru. Our intention is to aid the design of new RTI interventions or the translation of existing ones from high income nations to poorer ones based on locally relevant risk factors.Materials and MethodsStudy Design and SettingThis analysis is a sub-study of a large, community based cross sectional study of childhood injuries in San Juan de Miraflores (SJM), a poor, urban district of Lima, Peru. It includes results from the cross sectional study and two nested case control studies exploring personal and environmental risk factors for child pedestrian RTIs. Studies were conducted between January 2005 and July 2006.ParticipantsCross sectional and personal risk factor case control studiesIn the cross sectional study, six health promoters with high school graduate level education administered door to door surveys in 12 SJM zones, divided along existing neighbourhood borders. Staff began randomly and proceeded until each zone was completed. Households with a consenting adult and at least one resident child aged ≤18 were eligible.In the personal risk factor case control study, health promoters administered follow up surveys to cases of child pedestrian RTIs and randomly selected age and gender matched controls from the original study. Subjects were recruited to a goal of 100 cases and 200 controls. Cases were children who incurred a RTI during pedestrian activity in SJM from the year 2000 onward. RTIs occurring in parking lots, driveways, or while the vehicle was reversing were excluded. Two controls per case were selected by random assortment of all potential controls, and then random number generation to identify the first and subsequent controls. Controls were included if they could be age (within one year) and gender matched to a case, and there was no family history of pedestrian RTI. There was no compensation for participation.Environmental case control studyThe environmental case control study used the same case-control sets as in the personal risk factor study. Goal recruitment was 40 case and 80 control environments. Case sites of RTIs were included if the environment in which the RTI occurred had not changed since the time of the injury (as reported by the guardian of the injured child), and the RTI occurred between the hours of 6:00 and 20:00 (to minimize personal safety risk to staff as the district in which the study took place was quite dangerous). Control environments were selected by considering the origin and destination of the case child prior to injury, and the distance (number of blocks) from the origin that the RTI occurred. As an example, if a case was injured travelling from his local market to home, three blocks from the market, we then identified which market the control subject normally visited, and the route they would take from the market to home. The control site was then three blocks from their market, on their route home.Data CollectionAll study surveys were extensively pilot tested, and completed surveys were reviewed by the principal investigator (JD).Cross sectional and personal risk factor case control studiesIn the cross sectional study we administered a semi-structured survey including the core data sets recommended by the WHO [29]. Children aged ≥12 years and present at the time of the survey were interviewed in the presence of a guardian. If the child was not present or was <12 years of age, a guardian was interviewed. The personal risk factor study required that the injured child and guardian be present at the time of interview and collected demographic data and school information in relation to the year in which the injury (or case injury for controls) took place and precise RTI time and location data for cases.Environmental case control studyIn the environmental case control study, structured, 1.5 hour assessments were performed. Briefly, we evaluated: pedestrian movement, including volume, use of cross walks, and street vendor density; vehicle movement, including speed, vehicle specific volumes (cars, trucks, public transportation, motorcycle taxis), and traffic code infractions; pedestrian infrastructure, including sidewalks, crosswalks, and crossing lights; vehicle infrastructure, including road conditions, traffic lights, speed bumps, lane demarcation, and curb side parking.Environmental assessments were performed on the same day of the week and at the same time of day as when the case injury occurred. Case and control assessments were done simultaneously by two health promoters per site. Measurements were made only in the direction the vehicle was travelling prior to causing the case injury, on a section of road extending 150 meters from the injury site. All measurements were made by direct observation. Vehicle speed was recorded during a dedicated 30 minute period using digital timers to measure the time to traverse 150 meters.DefinitionsWe defined pedestrian activity as walking, running, or standing, but not cycling or skating. A road traffic injury (RTI) was any unintentional injury inflicted by a motorized vehicle. A serious RTI necessitated a healthcare consultation, i.e. visit to a hospital or health post. Of the RTIs identified in the cross sectional study, only serious RTIs were included in the case control studies. We considered poverty to be present if one or more of the following criteria were fulfilled: economic dependence (defined in Peru as ≥3 household occupants per wage earner), absence of indoor plumbing, dirt floors, and children in the home aged 6–12 not attending school. We considered overcrowding to be ≥4 people per room, excluding the kitchen, bathroom, and hallways. Education in the head of household and maternal education were low if primary schooling was incomplete. Years of residence refers to the total number of years in which the family has lived in the home where the interview took place. Speeds and volumes (i.e. pedestrian, vehicle) were considered high if they were in the highest tertile of recorded measurements. Avenues, streets, and roads, were defined according to city planning maps. Street vendors were sidewalk or street merchants without fixed locals.StatisticsSample size in the personal risk factor study was calculated with an assumed exposure in the control population of 50%, lowest detectable OR of 2.15, alpha equal to 0.05 and a power of 80 percent. Sample size in the environmental risk factor study was limited by time and funding considerations. Statistical analysis was performed using SPSS software (SPSS ®, SPSS Inc, ver. 11.5, Chicago, Illinois) and StatsDirect statistical software (StatsDirect Ltd ®, ver. 2.7.0, UK). Descriptive analyses determined the proportion and percentages of occurrences for binary and categorical variables. Conditional logistic regression methods were used to generate unadjusted matched odds ratios (OR) with 95% confidence intervals (CI) for each exposure variable and adjusted multivariate models for both personal and environmental risk factors. The best fitting multivariate models were built beginning with two variables with historic association to the outcome based on the review of the literature (poverty and maternal education for the personal risk factor study, and vehicle speed and volume for the environmental risk factor study), then adding subsequent variables if they improved the model. Improved models were those in which the −2 log likelihood ratio was greater than a critical value derived from the chi-square distribution table based on degrees of freedom at an alpha level of 0.05. Variables evaluated in the multivariate analysis but eliminated by statistical criteria are listed in Appendix S1. All statistical tests were two-sided and p-values <0.05 were considered statistically significant. Interactions between selected factors were tested however none were found to be significant.EthicsThe study protocols, informed consent and assent forms, and data collection instruments were approved by the human research ethics committee of Asociación Benéfica PRISMA (FWA 00001219). All interviewed children signed written assent forms and one guardian per household provided written consent for the cross sectional and case control portions of the study.ResultsDescriptive AnalysisParticipant recruitment and attrition are highlighted in Figure 1. In the cross sectional study, 21811 households were approached, of which 8039 were eligible for participation. Of these, 63% consented and were surveyed and the final analysis included 5061 households and 10210 children (median two per household; characteristics described in Table 1). Between 2000 and 2005, this population sustained 141 pedestrian RTIs, of which 117 (83%) were serious. No surveyed household reported child pedestrian RTI deaths during this period. In the personal risk factor study, there were four instances in which cases were clustered within households (two cases per household). Of these four homes, 50% (2·0) were poor, none had a low level of maternal education, there was a median of four resident children (range 2·0 to 5·0), and the median time of family residence was 11·5 years (range 2·0 to 15·0).10.1371/journal.pone.0003166.g001Figure 1In the cross sectional study, 24% (5832) and 36% (7940) of homes approached were not surveyed because no adult was home after two attempts or there were no resident children, respectively.Of the remaining 8039 households, 2973 (37%) refused to participate and five households (12 children) were excluded due to incomplete data. In the personal risk factor case control study, health promoters made two visits to homes in which no one was present, after which time the case or control was replaced. In 37 instances no adult was present on the repeat visit. Of the 40 cases selected for the environmental analysis, 93% were injured within the two years preceding the study. *Traffic volumes were too high to allow for accurate measurement in 2 case environments, thus they and their corresponding control environments were removed and replaced. RF = Risk Factors.10.1371/journal.pone.0003166.t001Table 1Cross sectional study population characteristics (n = 10210).CharacteristicNPercentGender-male526951·6Poverty- present563555·2Education of head of household-low*\n195119·1Overcrowding-yes7857·7Ages (years)†\n<14614·51–4229722·55–9286428·110–14279027·315–18179717·6*9 missing values.†1 missing value.Characteristics of serious pedestrian injuries are presented in Table 2. In the 1–4 and 5–9 year old groups, RTIs most commonly occurred during trips to or from the market (50% and 40%, respectively) and playing in the street (27% and 28%, respectively). In the 10–14 year old group, RTIs most commonly involved trips to or from school (28%), the market (23%) or a relative's home (23%). Younger children (aged 1–9) were more commonly injured while crossing in non designated areas (70% of RTIs in this age group).10.1371/journal.pone.0003166.t002Table 2RTI Characteristics (Cross Sectional Study).Characteristics of serious RTINPercentRTI requiring hospital/clinic visit*\n117<12 hours8676·1>12 hours2723·9Median days of hospitalization (n = 27)3\nAge at time of serious RTI (years)\n<10-1–42218·85–94740·210–143933·315–1897·7\nActivity\nMedian distance from home (blocks)2·5Median distance from point of origin (blocks)0·5RTI occurred during:Trip to/from store/market4135·0Trip to/from school1815·4Trip to/from relative's home1412·0Play2420·5Other†\n2017·1\nAlone or with another minor\n8270·1\nTime of day\nMorning3328·2Afternoon or evening8471·8\nLocation\nRoad or street4639·3Avenue7059·8Highway10·9\nAgent\nTaxi or mototaxi4841·0Private auto2723·1Public transportation (bus, minibus, or van)2622·2Other (truck, motorcycle)1613·7*4 missing values for duration of hospital visit; percentages shown are of the 113 with known duration.†Includes to/from friends home, bus stop, restaurant, parent's work, park, movies, and unspecified purpose.Of the 120 evaluated sites, only one had a traffic light, two had stop signs, and there were no posted speed limits. Tertiles derived from aggregate case and control data for pedestrian volume and vehicle speed and volume were used to define strata in the risk factor analyses. The middle pedestrian volume tertile was 101 to 201 pedestrians/hr, the middle vehicle speed tertile was 33.9 to 44.6 km/hr, and the middle vehicle volume tertile was 24 to 249 vehicles/hr. The median number of vehicles per hour at the case sites was 244·0 (range 0·0 to 4503·0), composed of motorcycle taxis (29%), public transportation (buses and vans- 28%), taxis (26%), and cars (15%). At control sites, the median number of vehicles per hour was 66·0 (range 0·0 to 1212·0), with a similar profile composed of motorcycle taxi (33%), public transportation (25%), taxi (25%), and cars (13%). The median vehicle velocity at case and control sites was 40·7 km/hr (range 18·2 to 60·5) and 39·8 km/hr (range 20·4 to 83·1), respectively.Risk Factor AnalysesUnivariate associations between case and control characteristics and pedestrian RTI are presented in Table 3. More hours per day in school was protective over pedestrian RTI, while larger streets, commercial or mixed commercial zones, and high vehicle volumes were associated with increased odds of pedestrian injury.10.1371/journal.pone.0003166.t003Table 3Characteristics of case and control participants and sites in the risk factor analyses*.CharacteristicCases (%)Controls (%)Matched OR (95% CI)\nPersonal risk factor analysis\n100200Gender- male67 (67·0)134 (67·0)NAAge- mean±SD11·5±4·411·9±4·1NAPoverty- present47 (47·0)103 (51·5)0·86 (0·52–1·41)Maternal education- low10 (10·0)9 (4·5)1·86 (0·73–4·75)Overcrowding- yes12 (12·0)12 (6·0)2·00 (0·90–4·45)Number of resident children- median (range)2·0 (1·0–9·0)2·0 (1·0–8·0)1·18 (0·99–1·42)Years of family residence- median (range)10·5 (0·1–46·0)14 (0·2–50·0)0·98 (0·96–1·00)Hours/day in school- mean±SD†\n\n4·8±0·96\n\n5·1±0·77\n\n0·56 (0·37–0·83)\nAttend school mostly in the afternoon- yes†\n27 (29·3)42 (21·8)1·37 (0·79–2·40)Walks to school- yes†\n83 (89·0)164 (85·0)1·36 (0·64–2·91)Allowed to play in street- yes†\n40 (40·0)67 (33·7)1·38 (0·82–2·32)Family has car- yes13 (13·0)38 (19·0)0·66 (0·35–1·27)Home with yard- yes66 (66·0)128 (64·0)1·09 (0·66–1·79)Blocks from home to nearest park- median (range)†\n1·0 (0·0–12·0)1·0 (0·0–10·0)0·98 (0·83–1·15)\nEnvironmental risk factor analysis\n4080Time of day- morning7 (17·5)14 (17·5)NALocation- avenue\n28 (70·0)\n\n35 (43·8)\n\n3·90 (1·51–10·09)\nZone- commercial or mixed commercial\n26 (65·0)\n\n35 (43·8)\n\n3·28 (1·24–8·65)\nPedestrian volume- high‡\n16 (40·0)24 (30·0)1·80 (0·79–4·09)Vehicle volume- high‡\n\n20 (50·0)\n\n20 (25·0)\n\n6·49 (1·83–23·0)\nVehicle Speed- high‡\n18 (45·0)22 (27·5)2·10 (0·95–4·63)Number of Street vendors- median (range)0 (0–25)0 (0–21)1·21 (0·99–1·48)Absent Lane Demarcation†\n29 (74·4)50 (62·5)1·71 (0·73–4·02)Dirt road- yes3 (7·5)13 (16·3)0·44 (0·12–1·59)Speed bump- yes2 (5·0)9 (11·3)0·44 (0·10–2·06)Gated community- yes2 (5·0)6 (7·5)0·67 (0·13–3·30)Pedestrian crosswalk present- yes2 (5·0)8 (10·0)0·50 (0·11–2·35)Sidewalk present- yes27 (67·5)62 (77·5)0·63 (0·28–1·42)>50% curbside parking4 (10·3)5 (6·3)2·38 (0·38–14·97)Park/play area nearby- yes17 (42·5)35 (43·8)0·96 (0·47–1·94)*Matched OR calculated with conditional logistic regression methods. OR denotes odd ratio, and CI confidence interval; NA- OR not calculated because cases/controls were matched by these variables.†For hours/day in school and attends school mostly in pm, and walks to school-7 cases and 7 controls were not attending school at the time of injury; For allowed to play in street- 1 missing control value; For blocks from home to nearest park- 4 missing control values; For absent lane demarcation- 1 missing case value.‡High designation corresponds to the highest tertile of all values recorded at all 120 sites (vehicle volume≥250 vehicles/hr; vehicle speed≥44·7 km.hr, pedestrian volume≥201pedestrians/hr).The final multivariate conditional logistic regression models for personal and environmental risk factors are shown in Table 4. More hours per day spent in school and years of family residency in the same home were protective against child pedestrian RTIs, while a greater number of children in the home, a greater number of street vendors, the absence of lane demarcations, and high vehicle volume and speed increased the odds of pedestrian injury.10.1371/journal.pone.0003166.t004Table 4Multivariate associations between personal and environmental risk factors and pedestrian RTI*.Risk FactorMatched OR (95%CI)\nPersonal\nPoverty0·58 (0·31–1·08)Low Maternal Education Level1·83 (0·65–5·17)Number of resident children\n1·25 (1·00–1·56)\n†\nYears of residence\n0·97 (0·95–0·99)\nHours/day in school‡\n\n0·52 (0·33–0·82)\nAttend school mostly in the afternoon‡\n1·31 (0·70–2·46)\nEnvironmental\nVehicle volume- high§\n\n7·88 (1·97–31·52)\nVehicle Speed- high§\n\n5·35 (1·55–18·54)\nNumber of Street vendors\n1·25 (1·01–1·55)\nAbsent Lane Demarcation∥\n\n6·59 (1·65–26·26)\n*OR denotes odd ratio, and CI confidence interval; Personal and environment al regressions were performed separately.†p = 0·048.‡7 cases and 7 controls were not attending school at the time of injury.§High designation corresponds to the highest tertile of all values recorded at all 120 sites (vehicle volume≥250 vehicles/hr; vehicle speed≥44·7 km.hr).∥1 missing case value.DiscussionEffective intervention design, or translation of existing interventions, for child pedestrian injury prevention to the world's poorer countries requires identification of locally relevant modifiable risk factors. This study describes the context in which child pedestrian RTIs occurred in an urban district of a major Latin American city, and identified both personal and environmental risk factors. Briefly, and in order of increasing strength of association, we found that more years of family residence in the same location and longer length of the school day were protective, while a greater number of children living in the home, a greater number of street vendors, higher vehicle speeds, absent lane demarcation, and higher vehicle volumes increased the risk of child pedestrian RTI. Our study of personal risk factors is among few such case control studies in Latin America, and, to the best of our knowledge, the environmental case control component is the first reported from the developing world.Personal risk factors for child pedestrian RTIs have been described extensively, but few are derived from case control studies in the developing world. We identified some similar associations, including familiarity with the local environment [20] (reflected by years of family residence) and the number of children living in the home [25]. However, other previously identified risk factors, such as poverty [15], [17], [20], [22], household crowding [12], [17], [20], [22], and low maternal education [20], [25] were not significant predictors within our population. SJM is a low income zone and the relative lack of economic variability may have weakened the effect of poverty. Overcrowding in this community may be compensated by the traditional Peruvian family dynamic in which multiple adult relatives live in the same or in neighbouring homes, increasing the number of caretakers per child. A similar effect may abrogate the anticipated importance of low maternal education. The protective effect of increased time spent in school has not been described previously. We found that each additional hour of school conferred a 48% decrease in the odds of a pedestrian RTI. This is unlikely a function of the added educational benefit of longer schooling, but rather a change in exposures. Children that are in school less may make more frequent trips to the market or spend more time playing in the street, the two most common exposures prior to RTI in this community. They may also be performing these activities at riskier times of day, when traffic patterns are more conducive to pedestrian RTI.Environmental case control studies, designed to identify environmental risk factors amenable to modification, are rarely performed in pedestrian RTI investigations. One reason is the challenge inherent in selecting appropriate control sites. Prior studies have first chosen a control child and then the control site in relation to this child's home based upon the distance and/or direction that the case RTI occurred in relation to the case home [16], [21]. This method, however, fails to consider two important variables - the points of origin and destination of the case child prior to injury. Our innovative technique for control selection used distance as in previous studies, but with relation to the points of origin and destination of the case child prior to the injury. Our approach takes account of what children are doing when they are injured and tries to answer the question of why only one of the three children doing the same thing in different places should suffer an injury. This was a challenging undertaking and case selection was limited to those for whom the journey's start and end points could be clearly defined (e.g. home to school) so that adequate definition of the comparable control journey was feasible. We found strong associations between child pedestrian RTIs and higher traffic volume and higher vehicle speed, consistent with other studies from wealthier countries [15], [16], [21], [23]. Similar to studies by Roberts and Mueller, our final model incorporates vehicle speed as a categorical, rather than continuous, variable [15], [21]. The risk of injury to a child may be similar on a street where the average vehicle speed is 25 km/hr (lowest tertile) and 35 km/hr (middle tertile), while the risk increases once the average vehicle speed reaches 45 km/hr (highest tertile). The risk of injury, however, on a street where the average vehicle speed is 55 km/hr may not be significantly different from that of the street where the average speed is 45 km/hr, as both these values are above the critical speed limit. The increased risk of pedestrian RTI associated with a greater number of street vendors and absent lane demarcation has not previously been described. Street vendors may create hazardous conditions by obstructing portions of the street, diverting traffic, concealing oncoming vehicles from view, or distracting pedestrians and drivers. Absent lane demarcations likely contribute to disorderly traffic flow, perhaps leading to unpredictable traffic patterns and making it more difficult for children to judge safe times to cross the street. These two factors are particularly common in the developing world and may be important considerations when designing child RTI interventions there.The strengths of this research include the large scale community based descriptive analysis and concurrent identification of both personal and environmental risk factors in the same community. The data are derived from and thus applicable to a developing world setting, addressing well recognized gaps in our understanding of child pedestrian RTIs in the world's highest risk regions. Finally, our novel approach to control environment selection makes an important contribution to the field of RTI research design. The low overall sampling rate of the cross sectional survey from which the cases and controls were generated should be considered when assessing the generalizability of the study. However, at the same time, the number of households and children surveyed was large. The difficult reality of conducting household surveys in a developing world setting is that childcare is often relegated to older siblings who cannot provide consent to participate and there exists a distrust of strangers grounded in the very tangible threat of home invasion and kidnappings. As only homes with at least one resident child were eligible for study inclusion, cases where an only child had been fatally injured in a RTI would have been missed, and, while likely a rare occurrence, this is a potential source of selection bias. Also, both case control studies involved injuries occurring up to five years prior to surveying, thus recall bias and changing roadside environments are important considerations. It is unlikely, however, that recall of socioeconomic and demographic factors would be biased significantly by having suffered an RTI. To minimize confounding by changing street environments, case families were asked if environmental modifications were made since the time injury. Affirmative or equivocal responses required selection of a new case site. Additionally, the sample size for the environmental risk factor study was small, thus increasing the probability of a type II error, and measurements were done by direct observation, therefore reported measures, while internally consistent and validated, should be considered approximations. Finally, children are only at risk for pedestrian RTIs when they are exposed to traffic while walking, therefore exposure factors (such time spent in school, walking to school, playing in the street, etc) are considered risk factors in our analysis. As the data is derived from case control studies, relationships between risk factors and outcomes represent associations and causality cannot be inferred.Our findings have important public health implications. Most injuries affected children aged 5–9 years, and occurred while unaccompanied, going to or from market, playing in the street, and crossing in non designated areas suggesting room for targeted behavioural interventions as well as the need for local traffic calming measures. Taxis and motorcycle taxis were the most frequent vehicles responsible, thus law enforcement could have a particularly helpful role. In particular, motorcycle taxis are a common mode of transport in Lima, but drivers are often under-aged, vehicles are often filled beyond capacity, and they frequently violate traffic codes. The protective effect of time spent at school suggests the viability of longer school days or increased access to after school programs as a means of preventing child pedestrian injuries. Finally, our environmental analysis provides evidence to support measures to reduce traffic volumes and speeds, limit the number of street vendors on a given stretch of road, and improve lane demarcation.It is clear that as disparities between high and low income countries continue to grow with regard to child pedestrian injuries, our goal should be to develop prevention strategies targeting risk factors relevant to a developing world context. This study advances that process by identifying both personal and environmental risk factors for child pedestrian RTIs in a major Latin America city.Supporting InformationAppendix S1(0.02 MB DOC)Click here for additional data file.\n\nREFERENCES:\n1. MurrayCJLopezAD\n1997\nAlternative projections of mortality and disability by cause 1990–2020: Global Burden of Disease Study.\nLancet\n349\n1498\n1504\n9167458\n2. PedenM\n2004\nThe world report on road traffic injury prevention\nGeneva\nWorld Health Organization\n3. World Health Organization\n1999\nInjury, a leading cause of the global burden of disease\nGeneva\nWHO\n4. AmeratungaSHijarMNortonR\n2006\nRoad-traffic injuries: confronting disparities to address a global-health problem.\nLancet\n367\n1533\n1540\n16679167\n5. NantulyaVMReichMR\n2002\nThe neglected epidemic: road traffic injuries in developing countries.\nBMJ\n324\n1139\n1141\n12003888\n6. HyderAAPedenM\n2003\nInequality and road-traffic injuries: call for action.\nLancet\n362\n2034\n2035\n14697797\n7. GargNHyderAA\n2006\nRoad traffic injuries in India: a review of the literature.\nScand J Public Health\n34\n100\n109\n16449050\n8. OderoWGarnerPZwiA\n1997\nRoad traffic injuries in developing countries: a comprehensive review of epidemiological studies.\nTrop Med Int Health\n2\n445\n460\n9217700\n9. RobertsI\n1995\nInjuries to child pedestrians.\nBMJ\n310\n413\n414\n10. RodriguezDYFernandezFJAcero VelasquezH\n2003\nRoad traffic injuries in Colombia.\nInj Control Saf Promot\n10\n29\n35\n12772483\n11. HijarMCKrausJFTovarVCarrilloC\n2001\nAnalysis of fatal pedestrian injuries in Mexico City, 1994–1997.\nInjury\n32\n279\n284\n11325362\n12. PlessIBPeckhamCSPowerC\n1989\nPredicting traffic injuries in childhood: a cohort analysis.\nJ Pediatr\n115\n932\n938\n2585230\n13. DuperrexOBunnFRobertsI\n2002\nSafety education of pedestrians for injury prevention: a systematic review of randomised controlled trials.\nBMJ\n324\n1129\n12003885\n14. ForjuohSN\n2003\nTraffic-related injury prevention interventions for low-income countries.\nInj Control Saf Promot\n10\n109\n118\n12772494\n15. MuellerBARivaraFPLiiSMWeissNS\n1990\nEnvironmental factors and the risk for childhood pedestrian-motor vehicle collision occurrence.\nAm J Epidemiol\n132\n550\n560\n2389759\n16. StevensonMRJamrozikKDSpittleJ\n1995\nA case-control study of traffic risk factors and child pedestrian injury.\nInt J Epidemiol\n24\n957\n964\n8557453\n17. RivaraFPGrossmanDCCummingsP\n1997\nInjury prevention. First of two parts.\nN Engl J Med\n337\n543\n548\n9262499\n18. RobertsI\n1993\nWhy have child pedestrian death rates fallen?\nBMJ\n306\n1737\n1739\n8363665\n19. PedenM\n2001\nProceedings of WHO meeting to develop a 5- year strategy for road traffic injury prevention\nGeneva\nWorld Health Organization\n20. AgranPFWinnDGAndersonCLDel ValleC\n1998\nFamily, social, and cultural factors in pedestrian injuries among Hispanic children.\nInj Prev\n4\n188\n193\n9788088\n21. RobertsINortonRJacksonRDunnRHassallI\n1995\nEffect of environmental factors on risk of injury of child pedestrians by motor vehicles: a case-control study.\nBMJ\n310\n91\n94\n7833733\n22. RivaraFPBarberM\n1985\nDemographic analysis of childhood pedestrian injuries.\nPediatrics\n76\n375\n381\n4034298\n23. RobertsIMarshallRLee-JoeT\n1995\nThe urban traffic environment and the risk of child pedestrian injury: a case-crossover approach.\nEpidemiology\n6\n169\n171\n7742404\n24. BruggeDLaiZHillCRandW\n2002\nTraffic injury data, policy, and public health: lessons from Boston Chinatown.\nJ Urban Health\n79\n87\n103\n11937618\n25. PlessIBVerreaultRTeninaS\n1989\nA case-control study of pedestrian and bicyclist injuries in childhood.\nAm J Public Health\n79\n995\n998\n2751039\n26. PedenMToroyanT\n2005\nCounting road traffic deaths and injuries: poor data should not detract from doing something!\nAnn Emerg Med\n46\n158\n160\n16046946\n27. PerelPMcGuireMEapenKFerraroA\n2004\nResearch on preventing road traffic injuries in developing countries is needed.\nBMJ\n328\n895\n28. BartlettSN\n2002\nThe problem of children's injuries in low-income countries: a review.\nHealth Policy Plan\n17\n1\n13\n29. World Health Organization\n2004\nGuidelines for conducting community surveys on injuries and violence\nGeneva"
+}

batch_11/PMC2529262.json

@@ -0,0 +1,4 @@
+{
+    "id": "PMC2529262",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2529262\nAUTHORS: Khaled Ezzedine, Thierry Simonart, Michel Candaele, Denis Malvy, Michel Heenen\n\nABSTRACT:\nWe present the case of an eighteen-year-old Caucasian white boy who was diagnosed with xeroderma pigmentosum type A at age 5 and who experienced over the past year disseminated small plaque psoriasis confirmed with skin punch biopsy. The psoriatic lesions were successfully treated with multipotent topical corticosteroids and systemic retinoids. To our knowledge, the association between psoriasis and xeroderma pigmentosum has not been previously reported and may be regarded as unlikely when considering the pathogenesis of both diseases.\n\nBODY:\nBackgroundXeroderma pigmentosum (XP) is a rare autosomal recessive disorder characterized by a defect of DNA-repair occurring during UV-induced damage. The disease is quite complex and different subsets of abnormalities in the DNA-repair system may be present during the course of the disease. Thus, patients with XP have a decreased cutaneous immune surveillance which results in an increased risk of UV-induced skin tumours at an early age. Basal cell carcinoma (BCC), squamous cell carcinoma (SCC), actinic keratoses, atypical moles and malignant melanoma, all associated with severe photoaging are commonly seen in such patients. The prognosis of XP is based on early diagnosis as to permit strict UV avoidance and early detection and treatment of skin tumours, especially in photo-exposed areas.Case presentationWe present the case of an eighteen-year-old white Caucasian boy who was diagnosed with XP type A at age 5. Other clinical signs include photophobia, keratitis and loss of eyelashes. Despite rigorous monitoring and photoprotection, the patient developed over 100 facial skin cancers (mainly BCC and SCC) treated with cryotherapy and surgery. Surprisingly, the patient experienced over the past year disseminated small plaque psoriasis (Fig. 1) confirmed with skin punch biopsy. S100 staining showed reduced density of Langerhans cells in healthy skin (Fig. 2), as previously reported in XP group A [1]. The psoriatic lesions were successfully treated with multipotent topical corticosteroids and systemic retinoids.Figure 1Association of small erythematous scaly plaques with multiple areas of hyperpigmentation resembling freckles on the patient's back.Figure 2S100 staining showing reduced density of Langerhans cells in healthy skin.DiscussionTo our knowledge, the association between psoriasis and XP has not been previously reported and may be regarded as unlikely when considering the pathogenesis of both diseases.XP patients exhibit Langerhans cell depletion, intensified impairment of natural killer (NK) cell function and enhanced UV-immunosuppression, possibly mediated through increased prostaglandin E2 production [1-4]. By opposition, psoriatic plaques are characterized by immune activation, with NK cell activation and decreased cyclo-oxygenase activity [5,6].In addition, in XP, unrestricted cellular proliferation is associated with inactivation of members of the iNK4a/Arf locus, such as p14 and p16 [7]. By opposition, in psoriasis, members of the iNK4a/Arf locus are overexpressed, which may contribute to the senescent switch and resistance of psoriatic plaques to cellular transformation despite altered differentiation, angiogenesis, increased telomerase activity, proliferative changes and apoptosis resistance characterising psoriatic skin [8,9].Further data are required to determine why and how those apparently antinomic diseases can coexist.AbbreviationsBCC: Basal cell carcinoma; DNA: desoxy ribonucleic acid; iNK: invariant natural killer; NK: natural killer; SCC: squamous cell carcinoma; UV: ultraviolet; XP: xeroderma pigmentosum.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsKE, TS, MH examined the patient. KE and TS were the major contributors in writing the manuscript. DM first seen the patient and helped to the redaction of the manuscript. MH and MC treated the patient and MH performed the histological examination for the skin biopsy. 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_11/PMC2529270.json

@@ -0,0 +1,4 @@
+{
+    "id": "PMC2529270",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2529270\nAUTHORS: Walter Zidek, Joachim Schrader, Stephan Lüders, Stephan Matthaei, Christoph Hasslacher, Joachim Hoyer, Peter Bramlage, Claus-Dieter Sturm, W Dieter Paar\n\nABSTRACT:\nBackgroundRecent clinical trials reported conflicting results on the reduction of new-onset diabetes using RAS blocking agents. Therefore the role of these agents in preventing diabetes is still not well defined. Ramipril is an ACE inhibitor (ACEi), that has been shown to reduce cardiovascular events in high risk patients and post-hoc analyses of the HOPE trial have provided evidence for its beneficial action in the prevention of diabetes.MethodsThe ADaPT investigation (\"ACE inhibitor-based versus diuretic-based antihypertensive primary treatment in patients with pre-diabetes\") is a 4-year open, prospective, parallel group phase IV study. It compares an antihypertensive treatment regimen based on ramipril versus a treatment based on diuretics or betablockers. The primary evaluation criterion is the first manifestation of type 2 diabetes. The study is conducted in primary care to allow the broadest possible application of its results. The present article provides an outline of the rationale, the design and baseline characteristics of AdaPT and compares these to previous studies including ASCOT-BLPA, VALUE and DREAM.ResultsUntil March 2006 a total of 2,015 patients in 150 general practices (general physicians and internists) throughout Germany were enrolled. The average age of patients enrolled was 67.1 ± 10.3 years, with 47% being male and a BMI of 29.9 ± 5.0 kg/m2. Dyslipidemia was present in 56.5%. 37.8% reported a family history of diabetes, 57.8% were previously diagnosed with hypertension (usually long standing). The HbA1c value at baseline was 5.6 %. Compared to the DREAM study patients were older, had more frequently hypertension and patients with cardiovascular disease were not excluded.ConclusionComparing the ADaPT design and baseline data to previous randomized controlled trial it can be acknowledged that AdaPT included patients with a high risk for diabetes development. Results are expected to be available in 2010. Data will be highly valuable for clinical practice due to the observational study design.\n\nBODY:\nBackgroundHypertension is the leading cause of morbidity and mortality worldwide [1]. The concomitant manifestation of type 2 diabetes mellitus leads to a substantial further increase in risk [2,3]. While about 50% of patients in German primary care were hypertensive in a recent cross-sectional survey, 12% of all patients had a co-manifestation of hypertension and diabetes [4].Not only hypertensive patients with diabetes, but also hypertensive patients without diabetes tend to be resistant to insulin stimulated glucose uptake and are hyperinsulinaemic compared with normotensive controls [5]. About 20% of patients with hypertension will develop type 2 diabetes in a three year period [6] and new onset diabetes in treated hypertensive patients is not trivial as recent studies suggest [7,8].The risk for subsequent cardiovascular (CV) disease in patients with pre-diabetes is not different from those who had both hypertension and diabetes already at baseline [9]. The adjusted relative risk of events was about 3-times higher in both previous and new onset diabetes compared to patients with hypertension but without diabetes [9].Antihypertensive drugs and new-onset diabetesThe roles of antihypertensive agents and in particular those that inhibit the RAS in the acceleration or deceleration of diabetes manifestation have been discussed controversial and study results on this question are not consistent.The RAS itself plays a pivotal role in the development of diabetes. Over-activity appears to be linked to reduced insulin and glucose delivery to the peripheral skeletal muscle and impaired glucose transport and response to insulin signalling pathways, thus increasing insulin resistance [10]. Activation of a local pancreatic RAS, in particular within the islets, may represent an independent mechanism for the progression of islet cell damage in diabetes. In fact, impaired pancreatic islet function may predominate quantitatively over peripheral insulin resistance in impaired glucose tolerance [11].Drugs that interrupt the RAS like angiotensin converting enzyme inhibitors (ACEi) and angiotensin receptors blockers (ARBs) are likely to be beneficial in the prevention of diabetes [10,12]. A series of recent large-scale prospective randomised studies of 3–6 year duration such as CAPP, INSIGHT, LIFE or ALLHAT, reported a remarkably consistent reduction in the incidence of type 2 diabetes in hypertensive patients reported with either ACEi-based or ARB-based therapy (reviewed by Jandeleit-Dahm in [10]). The comparator groups were based on thiazide-diuretics, β-blockers, the calcium channel blocker amlodipine or placebo, respectively.In a large meta-analyis, Abuissa et al. calculated the average risk reduction in 6 of these trials using ACEi and 7 trials using ARBs. The reduction of new onset-diabetes was 24% for ACEi, 23% for ARBs and 23% for the combined data-set [13]. Furthermore a recent network meta-analysis of randomized controlled trials showed that while patients taking betablockers and diuretics show an increased incidence of diabetes, it is reduced in patients using ACEi or ARBs (Figure 1) [14,15].Figure 1Diabetes incidence – results of full Bayesian network meta-analysis of 22 trials with 143153 patients [14], modified from [15].Endpoint studies to elucidate the role of antihypertensive agents on new onset diabetes related morbidity and mortality are however scarce. The VALUE trial with valsartan was the only trial to include new-onset diabetes as a pre-specified endpoint [16]. Patients were normoglycemic, those with abnormal glucose values were excluded. While 16.4% of patients in the amlodipine arm (up to 10 mg) developed diabetes over a mean follow-up of 4.2 years, 13.1% developed such in the valsartan arm (up to 160 mg); p < 0.0001. The ASCOT-BPLA study, which was a randomised controlled trial of the prevention of CHD and other vascular events by BP and cholesterol lowering in a factorial study design, was prematurely stopped in December 2004 [17,18]. The study was designed to resolve whether newer antihypertensive strategies that use calcium channel blockers (CCBs) and ACEi are superior to older treatments using betablockers and diuretics. As a key finding there was a substantial excess of new diabetes (increase of 30%) in the beta blocker/diuretic arm [17]. DREAM investigated the effect of ramipril (up to 15 mg) compared to placebo [6]. In this randomised controlled trial ramipril significantly increased regression to normoglycemia in patients with impaired glucose tolerance. It did however not influence the risk of a combined endpoint consisting of new-onset diabetes or death over a 3 year observational period. Interpretation of DREAM is limited by a number of details: 1) hypertension was not an inclusion criterion (mean blood pressure at baseline 136/83 mmHg), 2) comparison was made to placebo instead of diuretics or betablockers (which would be reasonable based on the analysis of Elliott [15]) and 3) betablockers were allowed in both the ramipril and placebo groups.Rationale for ADaPTThus, despite the strong evidence for a reduction of new-onset diabetes from several RCTs and meta-analyses, there is an ongoing controversy about the clinical significance, the comparability of agents within one drug class, or the generalisability of these findings into clinical practice [19-21]. The \"ACE inhibitor-based versus diuretic-based antihypertensive primary treatment in patients with prediabetes\" (ADaPT) study addresses this issue. On the basis of the existing body of evidence, it appeared likely that patients with impaired fasting glucose (IGF) according to the screening on pre-diabetes (PreDiSc Score) will benefit from tight blood pressure control and further effects from RAS-inhibition by the ACEi ramipril in terms of manifestation of type 2 diabetes. The long-term outcomes of this treatment regimen will be compared to a regimen based on diuretics and/or β-blockers.Materials and methodsDesignThe ADaPT investigation is a comparative Post Marketing Surveillance according to §67(6) German Drug Law, performed by the German Hypertension League. It was designed as an open, prospective, non-randomised parallel group observational investigation in 150 general practices (general physicians and internists) throughout Germany.Patient populationInclusion criteriaPatients eligible for this study were at high risk for the development of type 2 diabetes according to the modified PreDiSc Score [22]: They had to be ≥ 45 year old (amended, original protocol ≥ 55 years), have hypertension (systolic blood pressure ≥ 140 and/or diastolic blood pressure ≥ 90 mmHg), impaired fasting glucose (IFG) defined as glucose level 110–125 mg/dl in venous plasma or 100–109 mg/dl in capillary whole blood, and a glycosylated haemoglobin A1c (HbA1c) of 6–6.5% determined within the last six months.Exclusion criteriaPatients were excluded if they received any antidiabetic drug treatment, had overt type 2 diabetes, fasting blood glucose level ≥ 126 mg/dl in venous plasma or ≥ 110 mg/dl in capillary blood, or casual plasma glucose concentrations ≥ 200 mg/dl, congestive heart failure, chronic renal insufficiency, history of myocardial infarction, stroke, drug or alcohol abuse, or contraindications against one of the drugs applied.DefinitionsFor definitions of normal glucose tolerance (NGT), impaired fasting glucose (IFG), impaired glucose tolerance (IGT) and overt diabetes (DM) see Table 1. PreDiSc Score: The score indicates the presence of pre-diabetes with a diagnostic sensivity of 78% and a specificity of 37% using the following parameters: blood pressure (BP) ≥ 140/90 mmHg, capillary fasting blood glucose ≥ 100 mg/dl (STIX) and age ≥ 55 years. Sensivity can be increased to 79% and specificity to 74% by additional determination of the HbA1c value (≥ 6%) [22].Table 1Values of plasma glucose (venous blood) for the diagnosis of diabetes mellitus* and other categories of hyperglycemia according to DDG criteria [30]mg/dlmmol/lFasting2 h OGTTFasting2 h OGTTNGTNormal glucose tolerance< 100< 140< 5.6< 7.8IFGImpaired fasting glucose100–125-5.6–6.9-IGTImpaired glucose tolerance< 126and140–199< 7.0and7.8–11.0DMDiabetes mellitus≥ 126and/or≥ 200≥ 7.0and/or≥ 11.1IGT classification according to 2 h OGG is only appropriate if the NGT value is below the threshold value for diabetes mellitus.Antihypertensive treatmentPatients in Group 1 receive Ramipril either as monotherapy (Delix®, Sanofi-Aventis, Berlin) or in combination with Felodipin (Delmuno®, Sanofi-Aventis, Berlin), patients in Group 2 received any other diuretic-based or β-blocker-based therapy without using ACEi or ARBs. Ramipril was chosen as the antihypertensive drug in one group, as long-standing experience from several clinical studies including large endpoint studies have accumulated with this agent [23-25]. The Heart Outcomes Prevention Evaluation (HOPE) Study showed that ramipril is effective in preventing major cardiovascular events in high-risk patients without hypertension or those whose hypertension was sufficiently controlled with other treatments [26].Generally, treatment regimens in this study can be chosen in accordance with the recommendations of the German Hypertension League and the European Society of Hypertension [27,28] For initial treatment monotherapy or a low-dose combination regimen is suggested. If the response is inadequate, possible options include increasing the dose, changing the drug or introduction of further combination drugs [29]. All drugs are administered within the approved labelling.Advice about the investigation has been obtained by the institutional review board of the Charité Berlin, Germany – Campus Benjamin-Franklin. Written informed consent was obtained from every patient in writing. The planned follow-up period is four years.EndpointsThe primary evaluation criterion of this observational study is the first manifestation of type 2 diabetes according to the current guidelines of the German Diabetes Society (see table 1). [30] Further evaluation criteria are the deterioration of pre-diabetes indicated by an increase of HbA1c of at least 10% compared to baseline value within 4 years, the initiation of antidiabetic glucose lowering medication, an increase of fasting glucose levels, change of HbA1c compared to baseline, BP reduction, achievement of the target BP <130/80 mmHg after 12 months and at the 4 year follow-up, time needed to reach target BP, major cardiovascular (CV) events (first manifestation of symptomatic coronary heart disease (CHD) and/or peripheral arterial occlusive disease and/or cerebrovascular events), type and frequency of Adverse Events (AE) or Serious Adverse Events (SAE), and total mortality. In the diuretic-based therapy group, time to switch to ACE-based or ARB-based therapy will also be analysed.Investigational planTable 2 summarizes the investigational plan. Patients are seen at 7 scheduled visits. Vital signs (BP, heart rate) will be complemented by height and weight measurements (determinations of body mass index) and waist and hip circumference measurement at after 3, 6 and 12 months and thereafter at yearly intervals. Further, lab examinations of glucose, lipids, inflammatory (high sensitive C-reactive protein) and renal parameters (with cystatin C to assess renal function [30,31]) will be done in the same intervals. Ambulatory BP monitoring is facultative and will be performed in a subset of patients. AEs will be recorded and their severity, course and relation to the medication assessed by the treating physician. Prior as well as concomitant diseases and concomitant medication will also be assessed.Table 2ADaPT study planDocumentation/InvestigationBaseline12 wk6 mo1 yr2 yr3 yr4 yrClinical examination and medical historyxInformation on data protectionxBlood pressure, heart ratexxxxxxxPhysical examination: height, weight, waist and hip circumferencexxxxxLaboratory screening (central laboratory): Blood glucose, HbA1cxxxxxxxLaboratory values: hsCRP, total cholesterol, LDL-C, HDL-C, TG, cystatin C, potassium, albumin, OGTTxxxxxABPM (optional)xxxxxAE, SAExxxxxxWk = weeks; mo = months; yr = years; hsCRP = high sensitive C-reactive protein; ABPM = ambulatory blood pressure monitoring; AE = Adverse Events; SAE = Serious Adverse EventsStatistical Assumptions and sample size calculationSample size calculation for the primary endpoint was made under the assumption – based on the results of the ALLHAT study – that during the 4-year observational period 10.0% of patients in the ramipril-based antihypertensive regimen and 14.3% in the diuretic-based regimen will develop overt diabetes mellitus [21]. The detectable risk increase is compatible with 80% power and significance of 0.05 is 49% (OR 1,489). Based on this assumption, a sample size of n = 2001 was required. In terms of randomisation, addressing the literature evidence in favour of ACE inhibitor treatment, an imbalanced ratio of 2:1 for the number of patients in both treatment groups was chosen, resulting in a target inclusion number of 1334 patients in group 1 and 667 patients in group 2, respectively.Statistical analysesThe following parameters will be analysed: patient demographics including patient history, capillary und venous fasting glucose, HbA1c, BP, BMI, waist/hip ratio, concomitant medications, percentage of patients with prediabetes according to PreDiSc parameters [22], total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, urinary albumin, and high-sensitive C-reactive protein.Patients treated with ramipril (and ramipril-based combination therapy) and patients who received various other antihypertensive drugs (with the exception of ACEi or ARBs) will be compared. Statistical analyses of the data will be performed as exploratory analyses. Descriptive statistics for continuous target data per treatment group and per total contain the following: number of patients, means ± standard deviation, median, minimum and maximum. The absolute and relative frequency in percentages will be determined. Per treatment group 95% confidence intervals for the means of continuous target data as well as for the relative frequency of categorical target data will be calculated using appropriate methods.For comparison of the treatment groups with respect to the incidence of specific events (e.g. patients with first manifestation of diabetes mellitus type 2 or proportion of patients with deterioration of pre-diabetes), the chi-square or the log-rank tests will be used to compare the \"survival curves\". An interim analysis is scheduled after the first and second year of the observation. However, the overall significance levels will not be adjusted.Baseline CharacteristicsEnrolment in AdaPT started in August 2004. The last patient out of a total of 2,015 patients was included in March 2006 (table 3). 1,353 patients were enrolled into the ACEi-based group and 662 patients got a diuretic-based therapy. While age was similar between both groups the ACEi-based group had more male patients (51.4 vs. 42.6%). BMI (29.9 ± 5.0 vs. 29.8 ± 4.8 kg/m2) and waist-to-hip-ratio (0.95 ± 0.1 vs. 0.93 ± 0.1) were almost identical in both study groups. Dyslipidemia (56.5%), hypertension (57.8%) and overweight (43.3%) and obesity (42.6%) were the frequent baseline characteristics of patients in both groups. Baseline characteristics of AdaPT are displayed in table 3 and compared to other recent studies on the incidence of diabetes being treated with RAS blocking agents vs. conventional drugs.Table 3Baseline characteristics of the enrolled patients in AdaPT-study – compared to other trialsVALUE [16]ASCOT-BPLA [17]DREAM [6,38]AdaPTValsartanAmlodipineAmlodipineAtenololRamiprilPlaceboACEiDiuretic(n = 7649)(n = 7596)(n = 9639)(n = 9618)(n = 2.623)(n = 2.646)(n = 1.353)(n = 662)Age (mean, years)67.2 ± 8.267.3 ± 8.163.0 ± 8.563.0 ± 8.554.7 ± 10.954.7 ± 10.967.3 ± 10.466.5 ± 10.2Male gender (%)57.657.5777740.341.351.442.6BMI (mean, kg/m2)28.6 ± 5.128.7 ± 5.028.7 ± 4.628.7 ± 4.530.9 ± 5.630.9 ± 5.729.9 ± 5.029.8 ± 4.8 25–29.9 kg/m2 (%)n.a.n.a.n.a.n.a.n.a.n.a.43.742.6 ≥30 kg/m2 (%)n.a.n.a.n.a.n.a.n.a.n.a.42.443.2Waist-Hip-ratio Menn.a.n.a.n.a.n.a.0.96 ± 0.070.96 ± 0.070.99 ± 0.080.98 ± 0.07 Womenn.a.n.a.n.a.n.a.0.86 ± 0.080.87 ± 0.080.90 ± 0.090.90 ± 0.08History of Hypertension (%)92.7*92.0*81*81*34.443.556.859.8Heart rate (bpm)n.a.n.a.n.a.n.a.n.a.n.a.74.0 ± 9.473.0 ± 9.8Office systolic/diastolic BP (mmHg)154.5 ± 19.0/87.4 ± 10.9154.8 ± 19.0/87.6 ± 10.7164.1 ± 18.1/94.8 ± 10.4163.9 ± 18.0/94.5 ± 10.4136.1 ± 18.6/83.4 ± 10.8136.0 ± 18.1/83.4 ± 10.8147.4 ± 15.9/87.3 ± 9.3144.6 ± 15.3/86.5 ± 9.4HbA1c (mean %)n.a.n.a.n.a.n.a.n.a.n.a.5.6 ± 0.65.6 ± 0.7Risk factors Smoker (%)n.a.n.a.333244.145.015.613.6 Dyslipidemia (%)n.a.n.a.n.a.n.a.35.635.456.456.6 Hyperuricemia (%)n.a.n.a.n.a.n.a.n.a.n.a.21.723.0 MAU (%)n.a.n.a.n.a.n.a.n.a.n.a.6.25.4 CHD (%)45.646.0n.a.n.a.exclusion13.914.2 Stroke/TIA (%)19.819.8n.a.n.a.exclusionexclusionDrug therapy Aspirin or antiplatelet agents (%)n.a.n.a.191914.314.327.721.9 Thiazide diuretics (%)35.935.1n.a.n.a.9.510.00.40 Nonthiazide diuretics (%)n.a.n.a.5.95.600 ACEi41.341.4n.a.n.a.00.6 ARBs (%)10.710.6n.a.n.a.5.65.30.91.2 Betablockers (%)32.733.7n.a.n.a.17.217.50.30 CCBs (%)41.740.2n.a.n.a.12.812.918.615.3 Alphablockers (%)7.16.5n.a.n.a.1.92.22.12.0 Statins (%)n.a.n.a.11**10**12.413.519.417.4 Fibrates (%)n.a.n.a.2.12.30.10* previously treated for hypertension; n.a. = not available; BMI = body mass index; MAU = microalbuminuria; CHD = coronary heart disease; TIA = transitory ischemic attack; ACEi = ACE inhibitors; ARBs = Angiotensin receptor blockers; CCBs = calcium channel blockers** statins and fibrates combined.DiscussionAlthough there are several trials with a RAS based pharmacotherapy that report a reduction in the development of diabetes compared to diuretics and betablockers, these analyses were mostly post-hoc and not predefined. The only trials with a pre-defined new-onset diabetes endpoint were ASCOT-BPLA [17,18], VALUE [16] and DREAM [6] (new-onset diabetes as part of the primary endpoint). While there was a significant reduction of new-onset diabetes in ASCOT-BLPA (HR 0.70 [95%CI 0.63–0.78]) and VALUE (HR 0.77 [95%CI 0.69–0.86]) there was none in DREAM (HR 0.91 [95%CI 0.80–1.03]). Study duration was 5.5 (median), 4.2 (mean) and 3.0 (median) years. Within this setting the trial with the longest study duration had the most pronounced effect on diabetes development (see table 4).Table 4Design characteristics of AdaPT in comparison to other trialsVALUE [16]ASCOT-BPLA [17]DREAM [6,38]AdaPTValsartanAmlodipineAmlodipineAtenololRamiprilPlaceboACEiDiuretic(n = 7649)(n = 7596)(n = 9639)(n = 9618)(n = 2.623)(n = 2.646)(n = 1.353)(n = 662)Study designRCTRCTRCTObservational studyEndpointNew onset diabetes (secondary objective)New onset diabetes (tertiary objective)New onset diabetes or death (primary endpoint)New onset diabetes (primary evaluation criterion)Inclusion criteria Age (years)≥ 5040–79≥ 30≥ 45 Hypertension160–210/<115 mmHg≥ 160/100 mmHgn.a.≥ 140/90 mmHg Fasting plasma glucosenormalNormal110–125 mg/dl [6.1–7.0 mmol/l]110–125 mg/dl [6.1–7.0 mmol/l] Impaired glucose tolerancenormalNormal140–199 mg/dl [7.8–11.0 mmol/l]no inclusion criterionExclusion criteria Diabetesexclusion (for diabetes endpoint)exclusion (for diabetes endpoint)exclusionexclusion Cardiovascular diseasepossiblePossibleexclusionpossibleFollow-up (years)4.2 (mean)5.5 (median)3.0 (median)4 (planned)HR new-onset diabetes0.77 [95%CI 0.69–0.86]0.70 [95%CI 0.63–0.78]0.91 [95%CI 0.80–1.03]n.a.n.a. = not availableInclusion and exclusion criteria were also substantially different between ASCOT-BPLA, VALUE and DREAM. ASCOT-BPLA and VALUE included patients with treated or newly diagnosed hypertension being at least 40 years (ASCOT-BLPA) or 50 years old (VALUE). Neither impaired fasting plasma glucose nor glucose tolerance was an inclusion criterion. DREAM on the other hand included patients 30 years and above with either impaired fasting plasma glucose or glucose tolerance. Diagnosis of hypertension was not required.What is the additional value of AdaPT ?The primary goal of AdaPT is to compare the effects of two antihypertensive combination therapies, an ACE inhibitor based treatment with a diuretic- (or betablocker)-based treatment on the incidence of new-onset of type 2 diabetes. To provide adequate power to discriminate a potential differential effect of these therapies, the trial is being conducted in pre-diabetic patients with hypertension and metabolic disorders in which there is a high probability for the development of diabetes.AdaPT is conducted as an observational study in daily practice in Germany allowing for the widest possible applicability of the results obtained. This is important because there is considerable heterogeneity in patient management in daily practice and patients with pre-diabetes in clinical trials usually differ substantially from those in clinical trials. Taking the PreDiSc Score as a screening algorithm, the study allows physicians to effectively screen for a high risk for the development of diabetes. This would, in case of positive results, serve as an easy screening tool for high risk patients in the future.Compared to the previously mentioned trials AdaPT included patients at a higher risk for the development of diabetes based on the selection criteria age, presence of hypertension, an impaired fasting glucose and the missing exclusion of prior cardiovascular disease. As opposed to DREAM (Placebo control) AdaPT includes patients treated with betablockers or diuretics as a control group. This further enhances the likelihood of a differential effect based on the previously mentioned data by Abuissa [32], Elliott [15] and Lam [14] (see also Figure 1). Together with the 4 year follow-up it appears likely that AdaPT may document a reduced incidence of diabetes.The PreDiSc ScoreThe fact that a score (PreDiSc) is applied in the ADaPT investigation to prospectively identify eligible patients represents a novel approach, since it shifts the conventional focus from individual risk factors (hypertension, dyslipidaemia) to a more comprehensive view that considers absolute patient risk [3,33]. To our knowledge, such an approach has only been pursued in retrospective post-hoc investigations, for example in a current analysis of the LIFE study [34].The oral glucose tolerance test (OGTT) is the standard screening test in high risk populations (identified by medical history), but the fasting plasma glucose test is more convenient under daily practice conditions. [35] Determining fasting plasma glucose lacks sensitivity however and may miss a number of patients with diabetes. Another possible variable to determine glycaemic control is HbA1c but is less suitable for a general screening. [36,37]Against this background, the Pre-Diabetes Score (PreDiSc) Study established a set of easy-to-determine clinical and/or laboratory parameters with close correlation to the outcomes of an OGTT [22]. Indeed, using the HbA1c alone yielded low sensitivity (58%) as did fasting glucose alone (62%). However, a combination of age ≥ 55 years, systolic BP of ≥ 140 mmHg, fasting glucose ≥ 110 – 126 mg/dl, elevated HbA1c ≥ 6% and abdominal obesity (waist circumference > 88 cm in women and 102 cm in men) was associated with high sensitivity of IGT (i.e. identification of individuals with pre-diabetes: 79%) as well as high specificity (i.e. exclusion of individuals without pre-diabetes: 74%) [22]. The low acceptance of the OGTT and the non satisfying sensitivity and/or specificity of the sole HbA1c determination speak against these parameters as inclusion criteria for studies in daily practice. In contrast, the satisfactory predictive value of the clinically easy to determine dataset evaluated in PreDiSc was the rationale to use this score as screening procedure in a prospective study. Hypertensive patients fulfilling the PreDiSc criteria have a very high likelihood to progress to overt diabetes. Compared to the original PreDiSc score, on the basis of practical experiences in the initiation phase of the study two amendments to the ADaPT protocol became necessary: first, HbA1c was to be measured in a central laboratory instead of the originally foreseen local laboratories (due to wide variation in locally determined values). Second, the age criterion was reduced to 45 years or older in order to accelerate the inclusion rate.Strength and limitationsCertain limitations of the investigation have to be taken into account: first, it is controlled, but not randomised. This means that the comparability of both cohorts can be assessed retrospectively in terms of known factors that may influence the outcomes (e.g. age, gender, comorbidity), but not in terms of unknown bias. Second, the majority of patients will require combination therapy of two, three or even more antihypertensive drugs of various classes. At clinical practice conditions, a substantial proportion of patients may receive \"unallowed\" medications in the course of the investigation (i.e., from the regimen of the other arm). On the other hand, the real practice conditions in this investigation convey substantial benefits that will extend the knowledge from randomised studies. Patients will be less selected than typical study patients. The dosing regimens and the combinations will reflect current use (e.g. lower doses as compared to the US for some drugs, inclusion of ARBs, etc.) and thus the results can be easily extrapolated to day-to-day clinical use.ConclusionAlthough there are some published RCTs on the development of diabetes in patients on antihypertensive drugs, the results are inconclusive and require further investigation. The ADaPT study will provide important clinical data in a group of patients being at high risk to develop diabetes for which clear guideline recommendations regarding choice of antihypertensives are still missing.Conflict of interestDr. Paar declares to be an employee of Sanofi-Aventis Germany. All other authors have attended advisory boards and have held lectures for a number of pharmaceutical companies including Sanofi-Aventis.Authors' contributionsAll authors have made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data. PB has drafted the manuscript. The other authors revised the manuscript for important intellectual content. All authors have given final approval of the version to be published.\n\nREFERENCES:\nNo References"
+}

batch_11/PMC2529274.json

@@ -0,0 +1,4 @@
+{
+    "id": "PMC2529274",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2529274\nAUTHORS: Rajeshkumar U Koladiya, Amteshwar S Jaggi, Nirmal Singh, Bhupesh K Sharma\n\nABSTRACT:\nBackgroundStatins, HMG-CoA reductase inhibitors, are widely prescribed drugs for dyslipidemias. Recent studies have indicated number of cholesterol independent actions of statins including their beneficial effects on vascular endothelial dysfunction and memory deficits associated with dementia of Alzheimer's type. However the potential of statins in dementia of vascular origin still remains to be explored. Therefore, the present study has been designed to investigate the effect of Atorvastatin & Pitavastatin on vascular endothelial dysfunction associated memory deficits in rats. In this study L-Methionine induced vascular dementia was assessed by Morris water-maze (MWM) test. Biochemical analysis was also performed to unfold possible mechanism of statins mediated modulation of vascular dementia.ResultsL-Methionine produced endothelial dysfunction as reflected by significant decrease in serum nitrite concentration. L-Methionine treated rats performed poorly on MWM indicating impairment of memory as well. These rats also showed a significant rise in brain oxidative stress, acetylcholinesterase (AChE) activity and serum total cholesterol levels. Both Atorvastatin as well as Pitavastatin attenuated L-Methionine induced endothelial dysfunction associated memory deficits. Statins also reversed L-Methionine induced rise in brain oxidative stress, AChE activity and serum cholesterol.ConclusionThe beneficial effects of statins may be attributed to their multiple effects and the study highlights the potential of these drugs in vascular dementia.\n\nBODY:\nBackgroundDementia of vascular origin i.e. Vascular dementia (VaD) has gained much attention in the recent times. After Alzheimer disease (AD), VaD is the second most common cause of dementia. In the vascular system, nitric oxide (NO) generated by endothelial nitric oxide synthase (eNOS) plays an important role in maintenance of vascular tone [1]. Hyperhomocysteinemia (Hhcy), or elevation of plasma total homocysteine, is an important risk factor for cardiovascular disease, stroke and vascular dementia [2-4]. Hhcy has been shown to induce endothelial dysfunction by decreasing the bioavailability of NO, and increasing vascular oxidative stress [5]. The decreased NO level has been demonstrated to contribute to the pathogenesis of dementia [6].Increased levels of homocysteine have been documented to produce changes in structure and function of cerebral blood vessels along with oxidative stress, which play a key role in cerebral vascular dysfunction [7]. Oxidative stress and vascular dysfunction are recognized as important contributing factors in the pathogenesis of AD and other dementia of vascular origin [6]. In AD and other neurodegenerative diseases, structural deformities in the cerebral capillaries lead to impairment of cerebral perfusion with subsequent neuronal dysfunction and death [8]. The well established risk factors of endothelial dysfunction and subsequent vascular dementia such as hypertension, history of stroke, diabetes mellitus and hypercholesterolemia are all associated with high risk of AD. The noted vascular dysfunction (vascular deformities) in AD and common risk factor of AD and VaD suggest a great overlap between AD and vascular dementia [9]. Moreover, Hhcy has been documented to increase cholesterol synthesis [10]. Studies have revealed that in addition to elevated β-amyloid peptides and ApoE levels, high cholesterol level is another important risk factor for AD [11].Only limited therapeutic interventions are available to reduce the incidence of VaD. Cholinesterase inhibitors, calcium channel blockers and glutamate antagonists are few classes of pharmacological agents which are being clinically explored to reduce symptomatically the impact of cognitive dysfunction associated with vascular dementia [12]. However, an agent that should improve both endothelial dysfunction and associated dementia still need to be explored. Very recently, the focus has been directed towards statins (HMG-CoA reductase inhibitors), which are most widely prescribed drugs for dyslipidemias [13]. Statins in additions to their cholesterol lowering action are known to possess many cholesterol independent actions including favorable effect on vascular endothelium [14]. Moreover, there is an emerging data indicating that statins exert neuroprotective and antioxidant actions [14]. Statins have been shown to reduce the risk of ischemic stroke and related memory impairment by a variety of mechanisms [15]. Epidemiological studies have suggested that individuals above 50 years of age, who were receiving statins, had a substantially lowered risk of developing dementia, independent of the presence or absence of untreated hyperlipidemia, or exposure to non-statin lipid-lowering drugs [16]. However, there are conflicting observations regarding the effect of statins on cognitive functions. Although, there are a few studies showing cognitive decline [17], some studies showing no effect on memory [18,19], yet few studies suggest improvement of cognitive functions with statin therapy. Therefore, implication of statins in endothelial dysfunction and related dementia deserves further investigation.ResultsEffect of Vehicle/Atorvastatin/Pitavastatin/L-Methionine on escape latency time (ELT) and time spent in target quadrant (TSTQ), using Morris water maze (MWM)Vehicle treated (0.5%w/v CMC, 10 ml/kg/p.o.) rats showed a downward trend in their ELT. There was a significant (p < 0.01) fall in day 4 ELT, when compared to day 1 ELT of these rats (Table 1), reflecting normal learning ability. Further on day 5 a significant (p < 0.01) rise in TSTQ was observed, when compared to time spent in other quadrants (Figure 1), reflecting normal retrieval as well.Table 1Effect of Atorvastatin and Pitavastatin on L-Methionine induced changes in day 4 escape latency time (ELT), using Morris Water Maze.GroupsTreatmentDose (kg/day, p.o.)ELT (day 1) in secELT (day 4) in secIControl10 ml(0.5%w/w CMC)81.5 ± 4.520.2 ± 2.2aIIL-Methionine1.7 g93.8 ± 4.249.9 ± 2.4bIIIAtorvastatin per se10 mg85.5 ± 4.122.4 ± 3.4IVPitavastatin per se10 mg82.3 ± 4.321.3 ± 3.8VL-Methionine + Atorvastatin10 mg81.9 ± 3.927.0 ± 2.4cVIL-Methionine + Pitavastatin10 mg82.5 ± 4.427.9 ± 3.6cEach group (n = 10), represent mean ± SEM.a = p < 0.01 Day 1 Vs Day 4 ELT in vehicle control.b = p < 0.05 Vs Day 4 ELT in vehicle control.c = p < 0.05 Vs Day 4 ELT in L-Methionine treated group.Figure 1Effect of Atorvastatin and Pitavastatin on L-Methionine induced changes in day 5 Time Spent in Target quadrant (TSTQ), using Morris Water Maze. (L-Meth = L-Methionine; Atorva = Atorvastatin; Pitava = Pitavastatin). Each group (n = 10), represent mean ± SEM. a = p < 0.05 Time Spent in Q1, Q2, Q3, Quadrant Vs Q4 quadrant in Control. b = p < 0.05 Vs Time Spent in Target Quadrant Q4 of Control. c = p < 0.05 Vs Time Spent in Target Quadrant Q4 of L-Methionine treated.Administration of Atorvastatin (10 mg/kg/p.o., 2 weeks and 4 day)/Pitavastatin (10 mg/kg/p.o., 2 weeks and 4 day) did not show any significant per se effect on ELT and TSTQ as compared to vehicle treated rats. (Table 1 and Figure 1) L-Methionine (1.7 g/kg/p.o., 4 weeks and 4 day) administration produced a significant increase (p < 0.05) in day 4 ELT, when compared to day 4 ELT of vehicle control (Table 1) indicating impairment of acquisition. Further L-Methionine administration also produced a significant (p < 0.01) decrease in TSTQ, when compared TSTQ of vehicle control animals (Figure 1), indicating impairment of memory as well.Effect of Atorvastatin/Pitavastatin on L-Methionine induced impairment of learning and memory using Morris water mazeAdministration of Atorvastatin (10 mg/kg/p.o., 2 weeks and 4 day), Pitavastatin (10 mg/kg/p.o., 2 weeks and 4 day), significantly (p < 0.05) prevented L-Methionine induced rise in day 4 ELT, indicating reversal of L-methionine induced impairment of acquisition (Table 1). Further treatment of these drugs also attenuated L-Methionine induced decrease in day 5 TSTQ in a significant manner (p < 0.05), indicating reversal of L-methionine induced impairment of memory (Figure 1).Effect of Atorvastatin/Pitavastatin on L-Methionine induced change in endothelium dependent relaxationAcetylcholine (ACh) and sodium nitroprusside (SNP) in a dose dependent manner produced endothelium dependent and independent relaxation in phenylephrine (3 × 10-6 M) precontracted isolated rat aortic ring preparation. L-methionine (1.7 g/kg/p.o., 4 weeks and 4 day) administration significantly (p < 0.05) attenuated acetylcholine induced endothelium dependent relaxation (Figure 2), however it did not affect SNP induced endothelium independent relaxation (Figure 3). Treatment of Atorvastatin (10 mg/kg/p.o., 2 weeks and 4 day), Pitavastatin (10 mg/kg/p.o., 2 weeks and 4 day), significantly (p < 0.05) abolished the effect of L-Methionine on endothelial dependent relaxation. Further Atorvastatin and Pitavastatin did not show any per se effect on endothelium dependent relaxation.Figure 2Effect of Atorvastatin/Pitavastatin on L-Methionine change in endothelium dependent relaxation using isolated rat aortic ring preparation. (L-Meth = L-Methionine; Atorva = Atorvastatin; Pitava = Pitavastatin). Each group (n = 10), represent mean ± SEM. Responses are expressed as % of maximum contraction induced by phenylephrine (3 × 10-6 M). a = p < 0.05 Vs Control. b = p < 0.05 Vs L-Methionine treated group.Figure 3Effect of Atorvastatin/Pitavastatin/L-Methionine induced on sodium nitroprusside induced endothelium independent relaxation using isolated rat aortic ring preparation. (L-Meth = L-Methionine; Atorva = Atorvastatin; Pitava = Pitavastatin). Each group (n = 10), represent mean ± SEM. Responses are expressed as % of maximum contraction induced by phenylephrine (3 × 10-6 M).Effect of Atorvastatin/Pitavastatin on L-Methionine induced change in serum homocysteine levelAdministration of L-Methionine (1.7 g/kg/p.o., 4 weeks and 4 day), produced a significant (p < 0.01) increase in serum homocysteine, when compared to vehicle treated rats. Treatment with Atorvastatin (10 mg/kg/p.o., 2 weeks and 4 day)/Pitavastatin (10 mg/kg/p.o., 2 weeks and 4 day) produced a significant (p < 0.05) reduction of L-methionine induced rise in serum homocysteine level (Table 2). Further, Atorvastatin (10 mg/kg/p.o., 2 weeks and 4 day)/Pitavastatin (10 mg/kg/p.o., 2 weeks and 4 day) did not show any significant per se effect on serum homocysteine level (Table 2).Table 2Effect of Atorvastatin and Pitavastatin on L-Methionine induced changes in Serum homocysteine.GroupsTreatmentDose (kg/day, p.o.)Serum homocysteine (μM)IControl10 ml(0.5%w/w CMC)4.23 ± 0.15IIL-Methionine1.7 g20.8 ± 0.84aIIIAtorvastatin per se10 mg3.95 ± 0.21IVPitavastatin per se10 mg3.92 ± 0.23VL-Methionine + Atorvastatin10 mg12.9 ± 0.38bVIL-Methionine + Pitavastatin10 mg13.5 ± 0.34bEach group (n = 10), represent mean ± SEM.a = p < 0.01 Vs serum homocysteine level of Control.b = p < 0.05 Vs serum homocysteine level of L-Methionine treated group.Effect of Atorvastatin/Pitavastatin on L-Methionine induced change in serum nitrite levelAdministration of L-Methionine (1.7 g/kg/p.o., 4 weeks and 4 day), produced a significant (p < 0.01) decrease in serum nitrite, when compared to vehicle treated rats. Treatment with Atorvastatin (10 mg/kg/p.o., 2 weeks and 4 day)/Pitavastatin (10 mg/kg/p.o., 2 weeks and 4 day) prevented L-methionine induced decrease in serum nitrite level in a significant (p < 0.05) manner (Figure 4). Further, Atorvastatin (10 mg/kg/p.o., 2 weeks and 4 day)/Pitavastatin (10 mg/kg/p.o., 2 weeks and 4 day) did not show any significant per se effect on serum nitrite level (Figure 4).Figure 4Effect of Atorvastatin and Pitavastatin on L-Methionine induced changes in serum nitrite level. (L-Meth = L-Methionine; Atorva = Atorvastatin; Pitava = Pitavastatin). Each group (n = 10), represent mean ± SEM. a = p < 0.01 Vs serum nitrite of Control. b = p < 0.05 Vs serum nitrite of L-Methionine treated group.Effect of Atorvastatin/Pitavastatin/L-Methionine induced change in total serum cholesterol levelsAdministration of L-Methionine (1.7 g/kg/p.o., 4 weeks and 4 day) produced a significant (p < 0.05), increase in total serum cholesterol levels of animals, when compared to vehicle control. Treatment with Atorvastatin (10 mg/kg/p.o., 2 weeks and 4 day)/Pitavastatin (10 mg/kg/p.o., 2 weeks and 4 days) attenuated L-Methionine induced rise in total serum cholesterol levels in a significant (p < 0.05) manner (Figure 5). Furthermore Atorvastatin (10 mg/kg/p.o., 2 weeks and 4 day)/Pitavastatin (10 mg/kg/p.o., 2 weeks and 4 day) did not show any significant per se effect on total serum cholesterol levels (Figure 3), when compared to vehicle control group (Figure 5).Figure 5Effect of Atorvastatin and Pitavastatin on L-Methionine induced changes in serum total cholesterol level. (L-Meth = L-Methionine; Atorva = Atorvastatin; Pitava = Pitavastatin). Each group (n = 10), represent mean ± SEM. a = p < 0.05 Vs serum total cholesterol of Control. b = p < 0.05 Vs serum total cholesterol of L-Methionine treated.Effect of Atorvastatin/Pitavastatin on L-Methionine induced change in brain acetyl cholinesterase (AChE) activityAdministration of L-Methionine (1.7 g/kg/p.o., 4 weeks and 4 day) produced a significant (p < 0.05), increase in brain AChE activity, when compared to vehicle treated rats. Treatment with Atorvastatin (10 mg/kg/p.o., 2 weeks and 4 day)/Pitavastatin (10 mg/kg/p.o., 2 weeks and 4 day) significantly (p < 0.05), prevented L-Methionine induced rise in brain AChE activity. Further Atorvastatin (10 mg/kg/p.o., 2 weeks and 4 day)/Pitavastatin (10 mg/kg/p.o., 2 weeks and 4 day) did not show any significant per se effect on brain AChE activity (Figure 6).Figure 6Effect of Atorvastatin and Pitavastatin on L-Methionine induced changes brain acetylcholinesterase (AChE) activity. (L-Meth = L-Methionine; Atorva = Atorvastatin; Pitava = Pitavastatin). Each group (n = 10), represent mean ± SEM. a = p < 0.05 brain AChE activity of Control. b = p < 0.05 Vs brain AChE activity of L-Methionine treated.Effect of Atorvastatin/Pitavastatin on L-Methionine induced change in oxidative stress levels of brainAdministration of L-Methionine (1.7 g/kg/p.o., 4 weeks and 4 day), produced a significant increase (p < 0.05), in brain thiobarbituric acid reactive species (TBARS) level (Figure 7) and a decrease (p < 0.01), in the level of reduced form of glutathione (GSH) (Figure 8), when compared to vehicle treated rats; hence reflecting induction of oxidative stress. Treatment with Atorvastatin (10 mg/kg/p.o., 2 weeks and 4 day)/Pitavastatin (10 mg/kg/p.o., 2 weeks and 4 day) significantly (p < 0.05) prevented L-Methionine induced oxidative stress (Figure 7, 8). Further Atorvastatin (10 mg/kg/p.o., 2 weeks and 4 day)/Pitavastatin (10 mg/kg/p.o., 2 weeks and 4 day) did not show any significant per se effect on oxidative stress level (Figure 7, 8).Figure 7Effect of Atorvastatin and Pitavastatin on L-Methionine induced changes in brain thiobarbituric acid reactive species (TBARS) level. (L-Meth = L-Methionine; Atorva = Atorvastatin; Pitava = Pitavastatin). Each group (n = 10), represent mean ± SEM. a = p < 0.01 brain TBARS level of Control. b = p < 0.05 Vs brain TBARS level of L-Methionine treated group.Figure 8Effect of Atorvastatin and Pitavastatin on L-Methionine induced changes in brain reduced glutathione (GSH) level. (L-Meth = L-Methionine; Atorva = Atorvastatin; Pitava = Pitavastatin). Each group (n = 10), represent Mean ± SEM. a = p < 0.01 brain GSH level of Control. b = p < 0.05 Vs brain GSH level of L-Methionine treated group.DiscussionYoung male rats were employed in the present study, as it is reported that aging and consequent variation of estrogen in blood modulates the activity of endothelial nitric oxide synthase (eNOS), which further affects the function of vascular endothelium and memory [20,21]. Morris Water Maze test employed in present study is one of the most widely accepted models to evaluate learning and memory of the animals [22,23]. Nitric oxide (NO) synthesized in the endothelium and its levels get attenuated during endothelial dysfunction. Endogenously formed NO is highly unstable and gets converted to nitrate and nitrite [24]. Therefore, serum nitrite concentration has been employed as specific a marker of endothelial dysfunction [5].A significant decrease in escape latency time (ELT day 4) of control animals during ongoing acquisition trials denoted normal acquisition of memory and an increase in time spent in target quadrant (TSTQ), in search of missing platform during retrieval trial indicated, retrieval of memory. These results are consistent to our earlier findings and reports from other laboratory [13,25,26].L-Methionine treatment for 4 weeks, in the present study significantly raised serum homocysteine level, decreased serum nitrite levels and markedly attenuated acetylcholine induced endothelium dependent relaxation, therefore, reflecting endothelial dysfunction. Further L-methionine administration also produced a significant impairment of acquisition and retrieval of memory as reflected by decreased Morris water-maze performance. Moreover, an enhancement of brain acetyl cholinesterase (AChE) activity, increase in brain oxidative stress (as reflected by rise in brain TBARS and fall in GSH levels) and increase in serum total cholesterol levels were also observed. Recently, it has been reported that chronic experimental hyperhomocysteinemia produce cognitive dysfunction [27,28], and increase in brain AChE activity [29]. L-Methionine induced hyperhomocysteinemia is a well established model of experimental endothelial dysfunction [30,31]. Hyperhomocysteinemia has been reported to induce endothelial dysfunction by decreasing the bioavailability of NO and by increasing vascular oxidative stress [5]. Our observation also supports above contention, as a significant fall in serum nitrite levels along with rise in oxidative stress levels (increase TBARS and decrease GSH) were noted in L-methionine treated rats.The increased level of homocysteine has been reported to produce change in structure and function of cerebral blood vessels along with oxidative stress, which play a key role in cerebral vascular dysfunction [7]. Several lines of evidences have strongly advocated a direct relationship between vascular endothelial dysfunction and dementia better known as vascular dementia [32,33]. Cerebral vascular endothelial dysfunction has also been shown to enhance progression of dementia of Alzheimer disease (AD) [33]. Enhanced levels of brain AChE activity and oxidative stress have also been noted in patients suffering form dementia of AD and other dementias [34].Further hyperhomocysteinemia has also been shown to be neurotoxic, and the neurotoxicity may be due to over activation of N-methyl-D-aspartate receptors or by enhanced vulnerability of hippocampal neuron to excitotoxic insults and amyloid β-peptide toxicity [35,36]. Moreover, methionine rich diet in rats has been demonstrated to enhance cholesterol concentration in the plasma and liver [10]. Several studies have also revealed high serum cholesterol level as another important risk factor of AD [37]. Therefore L-Methionine induced memory dysfunction in the present study may be attributed to its multiple effects i.e. decrease in serum nitrite level (endothelial dysfunction), rise in oxidative stress level, enhancement of brain AChE activity, serum total cholesterol as well as direct neurotoxicity.In the present study, treatment with Atorvastatin and Pitavastatin significantly improved L-Methionine induced endothelial dysfunction manifested in the terms of endothelium dependent relaxation; increased serum nitrite levels, decreased serum homocysteine, and decreased oxidative stress (decrease TBARS and increase GSH). Statins, in addition to their cholesterol lowering action has been reported to exert number of cholesterol independent actions i.e. pleotropic actions. Statins have been demonstrated to enhance the expression of eNOS in human endothelial cells [38]. They have been known to activate Akt/protein kinase B, which subsequently activates eNOS [39]. Studies with Atorvastatin and Simvastatin have shown to inhibit the expression of prepro-endothelin (ET)-1 mRNA and reduce plasma ET-1 levels, endothelin being a potent vasoconstricting agent [40]. Furthermore, statins appear to inhibit the synthesis of isoprenoids, compounds that are required for the posttranslational modification of important signaling molecules such as Rho, Rac, and Ras [41]. Inhibition of Rho activation has been shown to increase endothelial NO production [38] and reduces ET-1 expression [40]. Recently, Atorvastatin has been documented to improve the function of endothelium by lowering the expression of p22phox and production of reactive oxygen species [30,42]. Further, in another recent report it has been indicated that hyperhomocysteinemia induce impairment of NO production through the modulation of Cav-1 expression associated with a loss of eNOS in caveolae [43]. Statins have been shown to prevent the expression of caveolin, a negative regulator of eNOS [44]. Therefore Atorvastatin and Pitavastatin induced improvement of endothelial dysfunction in our study may be attributed to their stimulatory effect on endothelial nitric oxide production and their antioxidative action.Further, administration of Atorvastatin/Pitavastatin in the present investigation also, reversed memory deficit induced by L-Methionine (hyperhomocysteinemia). Several recent clinical reports have suggested that the net brain cholesterol concentration is regulated by serum cholesterol level and there is a cross talk between the CNS and peripheral cholesterol pools [45,46]. Cholesterol turnover appears to play a crucial role in the deposition and clearance of amyloid peptide in brain and ApoE is a cholesterol transporting protein that is associated with amyloid deposits [47,48]. Further, serum cholesterol, atherosclerosis, apolipoprotein-E and AD all appear to be interconnected [49,50]. Studies involving cultured rat cortical neurons have revealed neuroprotective action of Atorvastatin against glutamate induced excitotoxicity [51].In our recent studies, we have reported that Atorvastatin, Simvastatin and Pitavastatin reversed memory deficits of rats and mice associated with dementia of AD type and memory improving effects of these statins mediated through their cholesterol dependent and in dependent actions [13,25]. Furthermore, statins have also been shown to exert beneficial effects in cerebral ischemia and stroke providing neuroprotection via enhancement of NO production [52].Many studies, in the recent years have implicated a vital role for NO in neurophysiological process of learning and memory [53]. Inhibition of NO system impaired memory in rats [54,55]. Whereas, stimulation of NO production improved cognitive functions in Alzheimer patients [56]. NO, donors like molsidomine reversed scopolamine induced amnesia in rats [57]. NO probably, acts as retrograde messenger in the formation of long term potentiation (LTP) at the molecular level of learning and memory processes [58]. Therefore, Atorvastatin and Pitavastatin in present investigation appear to reverse L-Methionine induced memory deficits via multiple actions viz; decrease in total cholesterol, brain oxidative stress (decrease TBARS and increase GSH) levels, AChE activity and increase in serum nitrite levels. Nevertheless, further studies incorporating female rats as well as other species of animals such as mice, using different memory model (other than Morris water-maze), duly supported by histopathology of brain tissue are required in order to support and extend potential of these statins in endothelial dysfunction related memory deficits.ConclusionIt may be concluded that chronic L-Methionine administration (hyperhomocysteinemia) has produced endothelial dysfunction along with impairment of learning and memory (vascular dementia). Atorvastatin and Pitavastatin exerted beneficial effects on endothelial dysfunction and related memory deficits by virtue of their cholesterol dependent as well as cholesterol independent actions. Perhaps this is the first report highlighting potential of Statins in L-Methionine induced endothelial dysfunction associated memory deficits.MethodsAnimalsAge matched (six months old) male Wistar Albino rats, weighing 150–200 g were employed in the present study. Animals were procured from Institute of Veterinary Science, Izat-Nagar Bareilly (U.P), India. Rats were provided standard laboratory feed (Kisan Feeds Ltd, Chandigarh, India) and tap water ad libitum and were exposed to 12 h light and 12 h dark cycle. The animals were acclimatized to the laboratory condition before experiments. The experimental protocol was duly approved by Institutional Animal Ethics Committee (IAEC) and care of the animals was taken as per guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), ministry of Environment and Forests, Government of India, (Reg. No. 107/1999/CPCSEA).Drugs and ChemicalsAtorvastatin was a gift from Ind swift Ltd., Mohali, Punjab, India. Pitavastatin was a gift from Zydus Research Center, Ahmedabad, Gujarat, India. All other reagents purchased from Merck limited, Mumbai, India, SD fine-chemicals limited, Mumbai, India, Loba Chem, Mumbai, India and Sigma-Aldrich, USA. Atorvastatin, Pitavastatin and L-Methionine were suspended in 0.5% w/v of carboxy methyl cellulose (CMC).L-Methionine Induced endothelial dysfunction and Vascular DementiaRats, were administered L-Methionine (1.7 g/kg/day, p.o.) for 4 weeks to produce hyperhomocysteinemia-induced endothelial dysfunction [30]. Assessment of vascular endothelial function was carried out by measuring acetylcholine induced endothelium dependent relaxation and sodium nitroprusside induced endothelium independent relaxation using isolated aortic ring preparation according to the method of Pieper [59] together with estimation of serum nitrite concentration.Body weight of rats was monitored weekly. After 4 weeks, rats were subjected to Morris water maze test for the evaluation of their memory status. The L-Methionine treatment was continued during acquisition trials on Morris Water Maze.Morris Water Maze TestMorris Water Maze (MWM) test was employed to assess learning and memory of rats [22,23]. The MWM procedure was based on a principle, where the animals were placed in a large pool of water, as animals dislike swimming, their tendency was to escape from the water being accomplished by finding an escape platform. MWM consisted of large circular pool (150 cm in diameter, 45 cm in height), filled to a depth of 30 cm with water at 28 ± 1°C. The water was made opaque with non-toxic white colored dye. The tank was divided hypothetically, into four equal quadrants with help of two threads, fixed at right angle to each other on the rim of the pool. A submerged platform (10 cm2), painted in white was placed inside the target quadrants of this pool, 1 cm below surface of water. The position of platform was kept unaltered throughout the training session. Each animal was subjected to four consecutive trials on each day with gap of 5 min. The rat was gently placed in the water of the pool between quadrants, facing the wall of pool with drop location changing for each trial, and allowed 120 sec to locate submerged platform. Then, it was allowed to stay on the platform for another 20 sec. If it failed to find the platform within 120 sec, it was guided gently onto platform and allowed to remain there for 20 sec. Escape latency time (ELT) to locate the hidden platform in water maze was noted as an index of acquisition or learning. Animal was subjected to four acquisition trials daily for four consecutive days. On fifth day, the platform was removed and each rat was allowed to explore the pool for 120 sec. Mean time spent in all four quadrants was noted. The mean time spent by the animal in target quadrant searching for the hidden platform was noted as an index of retrieval.Acquisition TrialEach rat was subjected to four trials on each day. A rest period of 5 min was allowed in between each trial. Four trials per day were repeated for four consecutive days. Starting position on each day to conduct four acquisition trials was changed as described below and Q4 was maintained as target quadrant in all acquisition trials. Mean escape latency time (ELT) calculated for each day during acquisition trials and day 4 ELT was used as an index of acquisition (table 3).Table 3Mean escape latency time (ELT) calculated for each day during acquisition trials and day 4 ELT was used as an index of acquisitionDay1Q1Q2Q3Q4Day2Q2Q3Q4Q1Day3Q3Q4Q1Q2Day4Q4Q1Q2Q3Retrieval TrialOn fifth day the platform was removed. Rat was placed in water maze and allowed to explore the maze for 120 sec. Each rat was subjected to four such trials and each trial was started from different quadrant. Mean time spent in all three quadrants i.e. Q1, Q2 and Q3 were recorded and the time spent in the target quadrant i.e. Q4 in search of missing platform provided an index of retrieval. The experimenter always stood at the same position. Care was taken not to disturb the relative location of water maze with respect to other objects in the laboratory serving, as prominent visual clues. All the trials were completed between 09.00 to 17.00 hrs in semi sound proof laboratory.Biochemical ParametersCollection of sampleThe animals were sacrificed by cervical dislocation; thoracic aorta and brain tissue were carefully removed. Thoracic aorta was used for endothelium dependent and independent relaxation, whereas brain tissue was subjected to various biochemical estimations.The removed brains were homogenized in phosphate buffer (pH 7.4, 10% w/v) using Teflon homogenizer. The clear supernatant, obtained after centrifugation at 3000 rpm for 15 min, was used to estimate acetyl cholinesterase (AChE) activity, thiobarbituric acid reactive species (TBARS), reduced glutathione (GSH) and protein content.Blood samples for biochemical estimation were collected just before sacrificing the rats. The blood was kept at room temperature for 30 min and then centrifuged at 4000 rpm for 15 min to separate serum. Serum was used to estimate serum homocysteine, serum nitrite concentration and total serum cholesterol.Estimation of serum homocysteineDetermination of homocysteine was carried out using HPLC (Varian Inc., CA, USA) attached with fluorescent HPLC detector according to the method of Dimitrova et al [60]. 100 μl of serum sample was added to 1.5 ml eppendorff containing 10 μl of water and 5 μl of n-amyl alcohol and was gently vortexed. 35 μl of sodium borohydrate reagent (35 μl of 1.43 M sodium borohydride in 0.1 M sodium hydroxide), 35 μl of 1 M of hydrochloric acid and 50 μl of 10 mM monobromobimane in 4 M sodium EDTA (pH 7) were added to eppendorff tube containing serum sample, capped, mixed and incubated at 42°C for 12 min. Then, it was cooled, vortexed and maintained at RT for 10 min. The sample was centrifuged at 12,200 g for 10 min to remove protein, acidic clear supernatant was separated and supernatant was adjusted to pH 4 using 25 μl of 2 M Tris-HCL. The sample was then centrifuged at 12,200 g for 1 min and 100 μl of supernatant was aliquoted for HPLC analysis.A fixed volume autosampler was used to inject 20 μl of sample into 4.6 × 250 mm RP8 ultrasphere column equipped with brownlee RP 18 new guard column. The solvent mixture in ratio of 94.75: 5: 0.25 of water: methanol: acetic acid was maintained at pH 3.4 using 5 M NaOH in pump A and 100% methanol was maintained in pump B. The injection rate of sample was maintained at 2 ml per min. HPLC detector was set with excitation wavelength at 390 nm and emission wavelength at 418 nm. The sensitivity range and rise time of detector were set at 0.1 sec and 2 sec respectively. The calibration curve for homocysteine as homocysteine-S-bimane was plotted using 100 μl quality control serum fortified with 10 μl homocysteine (1–1000 μM) solution. The data of calibration curve were regressed and the curve was used to calculate serum concentration of homocysteine.Rats with serum homocysteine levels of > 10 μM were considered to be hyperhomocysteinemic.Estimation of serum nitrite concentrationSerum nitrite concentration was estimated using method of Sastry et al. [24]. 400 μl of carbonate buffer (pH 9.0) was added to 100 μl of serum or standards sample followed by addition of small amount (~0.15 g) of copper-cadmium alloy. The tubes were incubated at room temperature for 1 h to reduce nitrate to nitrite. The reaction was stopped by adding 100 μl of 0.35 M sodium hydroxide. Following this, 400 μl of zinc sulfate solution (120 mM) was added to deproteinate the serum samples. The samples were allowed to stand for 10 min and then centrifuged at 4000 g for 10 min. Greiss reagent (250 μl of 1.0% sulfanilamide prepared in 3 N HCl and 250 μl of 0.1% N-naphthylethylenediamine prepared in water) was added to aliquots (500 μl) of clear supernatant and serum nitrite was measured spectrophotometrically (DU 640B Spectrophotometer, Beckman Coulter Inc., CA, USA) at 545 nm. The standard curve of sodium nitrite (5 to 50 μM) was plotted to calculate concentration of serum nitrite.Estimation of total cholesterolTotal serum cholesterol was estimated spectrophotometrically (DU 640B spectrophotometer, Beckman Coulter Inc., CA, USA) at 540 nm by CHOP/POD-phosphotungstate enzymatic method [61] using commercially available conventional diagnostic kit (Monozyme India ltd., Secundrabad).Estimation of brain acetyl cholinesterase (AChE) activityThe whole brain AChE activity was measured by the method of Ellman et al [62] with slight modifications [63]. This was measured on the basis of the formation of yellow colour due to the reaction of thiocholine with dithiobisnitrobenzoate ions. The rate of formation of thiocholine from acetylcholine iodide in the presence of brain cholinesterase was measured using a spectrophotometer. 0.5 ml of supernatant liquid of the brain homogenate was pipetted out into 25 ml volumetric flask and dilution was made with a freshly prepared DTNB {5,5'-dithiobis (2-nitro benzoic acid)} solution (10 mg DTNB in 100 ml of sorenson phosphate buffer, pH 8.0). From the volumetric flask, two 4 ml portions were pipetted out into two test tubes. Into one of the test tube, 2 drops of eserine solution was added. 1 ml of substrate solution (75 mg of acetylcholine iodide per 50 ml of distilled water) was pipetted out into both of the test tubes and incubated for 10 min. at 30°C. The solution containing eserine solution was used for zeroing the colorimeter and change in absorbance per min. of the sample was read spectrophotometrically (DU 640B spectrophotometer, Beckman Coulter Inc., CA, USA) at 420 nm. AChE activity was calculated using the following formula:R=δ O.D.×Volume of Assay (3 ml)E×mg of proteinWhere R = rate of enzyme activity in 'n' mole of acetylcholine iodide hydrolyzed/minute/mg proteinδO.D. = Change in absorbance/minuteE = Extinction coefficient = 13600/M/cmEstimation of brain thiobarbituric acid reactive species (TBARS) levelThe whole brain TBARS level was measured by the method of Ohokawa et al [64] with slight modifications. 0.2 ml brain homogenate was pipetted out in a test tube, followed by addition of 0.2 ml sodium dodecyl sulphate (SDS), 1.5 ml of 30% acetic acid (pH-3.5), 1.5 ml of 0.8% thiobarbituric acid (TBA) and make up the volume up to 4.0 ml with distilled water (DW). The test tubes were incubated at 95°C for 60 min., and then cool it. 1.0 ml of DW and 5.0 ml of n-butanol: pyridine (15: 1 v/v) mixture was added to the test tubes and centrifuged at the 4,000 × g for 10 min. The absorbance of developed colour in organic layer was Measured spectrophotometrically at 532 nm (DU 640B spectrophotometer, Beckman Coulter Inc., CA, USA). The absorbance from a standard curve generated using 1,1,3,3, tetra-methoxy propane as standard (range = 1 nmol – 10 nmol) was extrapolated.Estimation of brain reduced glutathione (GSH) levelThe whole brain GSH level was measured by the method of Beutler et al [65] with slight modifications. Tissue homogenate was taken and the proteins were precipitated with 10% w/v chilled trichloroacetic acid. Samples were kept in ice bath and were centrifuged after 30 min. at 1000 × g for 10 min. at 4°C. GSH levels were measured in the supernatant. 0.5 ml supernatant was mixed with 2.0 ml of 0.3 M disodium hydrogen phosphate solution and 0.25 ml of freshly prepared DTNB {5,5'-dithiobis (2-nitro benzoic acid)} solution (40 mg/100 ml in 1% w/v sodium citrate) was added just before measuring the absorbance spectrophotometrically at 412 nm (DU 640B spectrophotometer, Beckman Coulter Inc., CA, USA). Different concentration of GSH standard was also processed similarly to prepare a standard curve (5–50 μg) simultaneously. Results have been expressed as n mole of GSH/mg of protein.Estimation of brain total proteinFor the estimation of total protein in brain, method of Lowry et al [66] with slight modifications was used. 150 μL of supernatant was taken in a test tube, volume was made up to 1 ml with distilled water than 5 ml of Lowry's reagent (freshly prepared mixture of 1% w/v copper sulphate, 2% w/v sodium potassium tartrate and 2% w/v sodium carbonate in 0.1 N NaOH in the ratio of 1:1:98 respectively), was added and mixed thoroughly. Mixture was allowed to stand for 15 minutes at room temperature and then 0.5 ml of 1:1 v/v diluted Folin-Ciacalteu reagent was added. Contents were vortexed and incubated at 37°C for 30 minutes. Then absorbance was determined spectrophotometrically at 750 nm (DU 640B spectrophotometer, Beckman Coulter Inc., CA, USA) against suitably prepared blank. A standard curved using 25–200 mg of BSA was plotted. The amount of total protein was expressed in mg.Experimental DesignSix groups, each group comprised 10 albino Wistar rats, were employed in the present study.Group I (Vehicle treated control)Rats were administered 0.5% w/v CMC (10 ml/kg/day, p.o.) for 4 weeks and then subjected to MWM test. The vehicle was also administered 45 min before acquisition trial conducted from day 1 to day 4 and retrieval trial conducted on day 5.Group II (L-Methionine treated)In order to induce hyperhomocysteinemia, the rats were administered L-methionine (1.7 g/kg/day, p.o.) for 4 weeks and then subjected to MWM test. The treatment of L-Methionine was continued (administered 45 min before) during acquisition trial conducted from day 1 to day 4. The animals were administered vehicle (0.5%w/v CMC, 10 ml/kg, p.o.) 45 min before retrieval trial conducted on day 5.Group III (Atorvastatin per se)Rats were administered Atorvastatin (10 mg/kg/day, p.o.) for 2 weeks and then subjected to MWM test. The treatment was continued (administered 45 min before) during acquisition trial conducted from day 1 to day 4. The animals were administered vehicle (0.5%w/v CMC, 10 ml/kg, p.o) 45 min before retrieval trial conducted on day 5.Group IV (Pitavastatin per se)Rats were administered Pitavastatin (10 mg/kg/day, p.o.) for 2 weeks and rest of protocol was same as mentioned in group III.Group V (L-Methionine + Atorvastatin treated)The hyperhomocysteinemic rats were treated with Atorvastatin (10 mg/kg/day, p.o.) for 2 weeks (3rd and 4th week of L-Methionine administration) and then subjected to MWM test. The co-administration of Atorvastatin and L-Methionine was continued (administered 45 min before) during acquisition trial conduct from day 1 to day 4. The animals were administered vehicle (0.5%w/v CMC, 10 ml/kg, p.o) 45 min before retrieval trial conducted on day 5.Group VI (L-Methionine + Pitavastatin treated)The hyperhomocysteinemic rats were treated with Pitavastatin (10 mg/kg/day, p.o.) and subjected to MWM test as described in group VI.Statistical analysisThe results were expressed as mean ± standard error of means (S.E.M.) The data for isolated aortic ring preparation was statistically analyzed using repeated measure ANOVA followed by Newman-Keul's test. Rest of the data obtained from various groups was statistically analyzed using one-way ANOVA followed by Tukey's Multiple Range test. The p < 0.05 was considered to be statistically significant.Authors' contributionsRUK carried out surgical operations, behavioral tests, and biochemical tests the data analysis in animal studies. BKS assisted in carrying out surgery, behavioral tests, and biochemical tests. ASJ carried out data analysis and participated in critical intellectual discussion and designing of the experiments. NS conceived the idea, coordinated the study, carried our data interpretation and drafted the manuscript. All authors read and approved the final manuscript.\n\nREFERENCES:\nNo References"
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+{
+    "id": "PMC2529276",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2529276\nAUTHORS: Sarah E Rosenbaum, Claire Glenton, Jane Cracknell\n\nABSTRACT:\nBackgroundEvidence-based decision making relies on easy access to trustworthy research results. The Cochrane Library is a key source of evidence about the effect of interventions and aims to \"promote the accessibility of systematic reviews to anyone wanting to make a decision about health care\". We explored how health professionals found, used and experienced The Library, looking at facets of user experience including findability, usability, usefulness, credibility, desirability and value.MethodsWe carried out 32 one-hour usability tests on participants from Norway and the UK. Participants both browsed freely and attempted to perform individually tailored tasks while \"thinking aloud\". Sessions were recorded and viewed in real time by researchers. Transcriptions and videos were reviewed by one researcher and one designer. Findings reported here reflect issues receiving a high degree of saturation and that we judge to be critical to the user experience of evidence-based web sites, based on principles for usability heuristics, web guidelines and evidence-based practice.ResultsParticipants had much difficulty locating both the site and its contents. Non-native English speakers were at an extra disadvantage when retrieving relevant documents despite high levels of English-language skills. Many participants displayed feelings of ineptitude, alienation and frustration. Some made serious mistakes in correctly distinguishing between different information types, for instance reviews, review protocols, and individual studies. Although most expressed a high regard for the site's credibility, some later displayed a mistrust of the independence of the information. Others were overconfident, thinking everything on The Cochrane Library site shared the same level of quality approval.ConclusionParadoxically, The Cochrane Library, established to support easy access to research evidence, has its own problems of accessibility. Health professionals' experiences of this and other evidence-based online resources can be improved by applying existing principles for web usability, prioritizing the development of simple search functionality, emitting \"researcher\" jargon, consistent marking of site ownership, and clear signposting of different document types and different content quality.\n\nBODY:\nBackgroundThe value of evidence-based medicine (EBM) – using updated, relevant and trustworthy evidence to inform medical decisions is widely acknowledged [1]. Recently the British Medical Journal nominated EBM as one of the 15 most important milestones in medicine since 1840 [2]. Easy access to high quality research has the potential to improve patient care, but there are obstacles that face health professionals attempting to use evidence in their practice. In an Australian survey, physicians identified insufficient time (74%), limited search skills (41%) and limited access to evidence (43%) as impediments to making better use of research data [3].Systematic reviews directly address several of these barriers, as their summarized form reduces the amount of time and search skills needed to access and appraise many individual studies [4]. A systematic review is a summary of individual studies addressing a clearly formulated question that uses systematic and explicit methods to identify, select, and critically appraise the relevant research, and to collect and analyse data from the included studies. The Cochrane Collaboration is an international organisation of volunteers dedicated to producing systematic reviews of rigorous methodological quality. These reviews are published in one of the databases on The Cochrane Library [5], a web site that has the potential to further simplify the task of finding trustworthy evidence. Additionally the Library hosts other databases for systematic reviews, health technology assessments and randomized controlled trials, making it a central online collection of varying types of evidence from a variety of sources.Part of the mission of The Cochrane Collaboration is \"to promote the accessibility of systematic reviews to anyone wanting to make a decision about health care\". The organization also aims to produce reviews that are easy to read and understand by someone with a basic sense of the topic [6]. But does the Library web site support the Collaboration's goals of clarity and ease of use, as well as the overreaching mission of making evidence accessible for decision making? We wanted to explore this question through observing the experiences of health professionals using The Cochrane Library. We were interested not only in site-specific problems but also in issues that might be relevant to other web sites publishing collections of evidence-based content.User experienceUsability testing is a method that is widely used in the field of web design to uncover errors and areas of improvement by observing users solving given tasks on the site [7,8]. There is increased recognition of the limitations of examining only task-related problems when attempting to understand why users' interactions with web sites might succeed or fail. Attention to the user's whole experience has begun to gain ground in the field of human-computer interaction [9]. Morville's \"honeycomb\" model (see Figure 1) distinguishes between seven separate facets of user experience, including findability, accessibility, usability, usefulness, credibility, desirability and value [10].Figure 1The honeycomb model of user experience, reproduced here with permission from Peter Morville, Sematic Studios LLC.A brief explanation of these terms:Findability: can users locate what they are looking for?Accessibility: are there physical barriers to actually gaining access, also for people with handicaps?Usability: how easy and satisfying is this product to use?Usefulness: does this product have practical value for this user?Credibility: is it trustworthy?Desirability: is it something the user wants? Has a positive emotional response to?Value: does this product advance the mission of the organization behind it?Our study aimed to explore the user experience of health professionals trying to find evidence in The Cochrane Library, building on methods from usability testing. In this article we use the honeycomb model to organize findings from our study to illustrate more general potential pitfalls and challenges particular to evidence-based online resources. At the end we suggest some guidelines for designers, writers and developers working to improve the user experience of these types of sites.MethodsWe carried out two series of user tests in 2005 (Test 1) and 2006 (Test 2), with participants from Norway and UK. The publisher of the site, Wiley-Blackwell, made changes to the site after Test 1, partly based on the results we uncovered. Most of these changes regarded branding at the top of the site, making The Cochrane Library the prominent identity and toning down the logo and universal navigation of the publisher. Therefore we altered the interview guide of Test 2 in small ways so that the questions would match the changes that had been made. See Additional file 1 for the complete interview guide we used in Test 2.We limited our selection to health professionals who used the Internet and had some knowledge of systematic reviews, to ensure that the results of the interface testing would not be confounded by unfamiliarity with the media or the site's content. We sent email invitations to lists of previous attendees of evidence-based practice workshops, employees in the Directorate of Health and Social Affairs in Oslo and individuals in evidence-based health care networks in Oxford. Volunteers who responded were screened by phone or email to assess whether they fitted the requirements, and also to find relevant topics of interest so that we could individually tailor test questions. We also asked them about their online searching habits, and what sources of online information they usually used in connection with work. We did not reveal the name of the site we were testing during recruitment. Test persons were promised a gift certificate worth the equivalent of $80 USD or a USB memory stick if they showed up for the test.Tests were performed individually and took approximately one hour. The test participant sat at a computer in a closed office together with the test leader who followed a semi-structured test guide. We recorded all movement on the computer desktop through use of Morae usability test software [11] and video-filmed the participant, who was prompted to think out loud during the whole session. We projected the filming of the desktop and the participant as well as the sound track, to another room where two observers transcribed, discussed, and took notes.The data was anonymous to the degree that participants' names were not connected to video, audio or text results. We received written permission to store the recordings for five years before deleting it, guaranteeing that video/audio tapes would not be used for any purpose outside of the study and not be published/stored in places of public access. The protocol was approved by the Norwegian Social Science Data Services and found in line with national laws for privacy rights.We began the test with preliminary questions about the participant's profession, use of Internet, and knowledge of The Cochrane Library. We then asked the participant to find specific material published on the Library starting from an empty browser window. Once on the site, we asked about their initial reactions to the front page, and they were invited to browse freely, looking for content of interest to themselves. Then we asked them to perform a series of tasks, some of which involved looking for specific content about topics tailored to their field or professional interests. For instance, a midwife was asked to find:- all information on the whole library that dealt with prevention of spontaneous abortion- a specific review about the effect of caesarean section for non-medical reasons- all new Cochrane Reviews relevant to the topic \"music used to relieve pain\".Other general tasks included finding help, finding the home page, and finding information about Cochrane. We also had specific tasks leading to searching and to reading a review. At the end, we asked if they had any general comments to the site and suggestions to how it could be improved.Our analysis was done in two phases. The aim of the first analysis was to provide the stakeholders and site developers with an overview and a prioritizing of the problems we had identified. At least two of us carried out content analysis of the transcripts, independently coding each test. These codes were then compared, discussed and merged. The topics were then rated according to the severity of the problem for the user. We rated severity in three categories: high (show-stopper, leads to critical errors or hinders task completion), medium (creates much frustration or slows user down), or low (minor or cosmetic problems).The second analysis was done to lift more generalizable issues underlying this article out of the site-specific data. We re-sorted the findings into the seven user-experience categories from the honeycomb model by re-reading the transcript, checking the context where the problems came from, and evaluating which of the seven categories best fit each finding. Severity-of-problem ratings from the first analysis were kept in the second analysis.We did not evaluate accessibility (the degree to which the website complied with standards of universal accessibility, for instance as defined by the Web Accessibility Initiative [12]), since user testing methods are not an effective way of gathering data on various aspects of this issue.The findings presented here are a selection of issues that received a high degree of saturation in our tests, and that we judge to be critical (\"high severity\") to the user experience of evidence-based web sites in general. This judgement is based on basic principles for web usability [7,13-15] as well as the principles underlying evidence-based health care: to successfully search for, critically appraise and apply evidence in medical practice [16].Most of the findings here are still of relevance to The Cochrane Library in its current format, though we have included some observations of problems that are now resolved, because they illustrate issues that are potentially important for others. Our aim is not to write a critical review of the library, but to highlight issues we found that can be important to user experience of evidence-based web sites for health professionals.ResultsParticipant profilesWe tested a total of 32 persons (See Table 1 for participant details). Test 1 included 13 persons from Norway, and Test 2 included five persons from Norway and 14 from the UK. Twenty-one of the 32 participants were non-native English speakers accustomed to reading in English.Table 1Participant detailsGenderAgeProfessionInternet use: FrequencyNative languagePlace of residence1F44MidwifeDailyNorwegianOslo2F43Sociologist, advisor in health-related govt. institutionDailyNorwegianOslo3F53Physical therapist/teacher1–2 times a monthNorwegianOslo4F45Midwife/researcherDailyOther (not English)Oslo5F-advisor in health-related govt. institutionUp to 5 times a weekNorwegianOslo6F-Masters in nursing science, lectures at college levelDailyNorwegianOslo7F39Midwife/teacherDailyNorwegianOslo8M49Medical Doctor/dept. director at health-related govt. institutionDailyNorwegianOslo9F28Psychologist at health station for youthNorwegianOslo10M40–50Medical Doctor/senior advisor at health-related govt. institutionDailyNorwegianOslo11F56Sociologist/Masters in health admin./advisor at health-related govt. institutionAlmost everydayNorwegianOslo12M25–35Physical therapistDailyNorwegianOslo13F28Physical therapist at county health stationUp to 5 days a weekNorwegianOslo14M43Psychologist at hospitalDailyNorwegianOslo15F34Medical Doctor at hospitalUp to 5 days a weekNorwegianOslo16M49Medical Doctor at hospitalDailyNorwegianOslo17F54Midwife/teacher3 times a weekNorwegianOslo18F23Nurse (recently graduated)3 times a weekNorwegianOslo19F42Research nurse5–10 hours a weekDanishOxford20F-Pediatric Nurse10–20 hours a weekEnglishOxford21F45Consultant, public health. Clinical dentist, doing an Mba10–20 hours a weekEnglishOxford22M35Medical Doctor10–20 hours a weekEnglishOxford23F31Psychiatrist10–20 hours a weekEnglishOxford24F46General practitioner20–40 hours a weekEnglishOxford25F41Mental Health nurse5–10 hours a weekEnglishOxford26M66Consultant Dentist Public HealthLess than 5 hours a weekEnglishOxford27F32Nursing, Post-doc in nursing-related field10–20 hours a weekEnglishOxford28F40Clinical orthodontistUp to 5 times a weekEnglishOxford29F45Occupational therapistLess than 5 times a weekOther (not English)Oxford30F50Nursing, Midwife, starting PhdUp to 5 times a weekEnglishOxford31M-DentistDailyEnglishOxford32M54General practitioner5–10 hours a weekEnglishOxfordParticipants were educated in nursing/midwifery (10); medicine (8); dentistry (4); physiotherapy (4); social sciences (3); psychology (2); and occupational therapy (1). They were currently working as health professionals in primary or secondary care (17); as government advisors working with health-related issues (7); as teachers at nursing/physiotherapy schools or universities (4); as research nurses (3); or as an editor for a patient information website (1).Most used the Internet daily or several times a week, and much of this use was work-related. All had searched the Internet for health-related information or evidence. Most participants reported that they normally looked for information in response to a specific problem. A few of them had strategies to keep up to date within a certain field on a more regular basis. When in need of information, the most common sources mentioned were colleagues, research databases, and the Internet. All but one participant had some previous knowledge of The Cochrane Collaboration and 25 of the 32 participants could provide at least a basic description of the term \"systematic review\". Twenty-six said that they had visited The Cochrane Library site previously.The findings that we included in this article are listed in Table 2.Table 2Main findings, sorted into the facets of the honeycomb user experience modelFindabilityDifficulty finding the web site through Google or other external searchDifficulty finding specific content on the site, using on-site search- non-English participants spelled search queries wrong- search engine too sensitive- keywords search didn't work properly- simple search produced unexpected results (i.e.: too few or too many of wrong type)- search results were misinterpreted, users confused document types- confusion when retrieving only a small number of search resultsTopics navigation not used or not seenMinimum of browsing even when encouraged to look around the siteUsabilityUnfamiliar language/jargon caused confusionText too smallToo dense, too much text (front page, Help, More information pages)Important content too far down on page (review pages)Not interested in reading whole reviewForrest plots unfamiliar and not intuitively locatedCredibilityUsers trusted content in The Cochrane LibraryConfusion about site ownership/neutrality due to dominance of publisher identity and universal navigation, weakens trustMisunderstanding about editorial quality evaluation – thinking all content on the whole site content has been reviewed by CochraneUsefulnessAssuming the library only dealt with medical topics (and not topics such as dentistry, nutrition, acupuncture)Misunderstanding targeted texts on front page, thinking content would be tailored for these groupsPerceived as an academic resourcePlain language summaries appreciatedDesirabilitySite seemed off-putting, overwhelmingSite can be alienating (research/academic identity and language)ValueFelt Cochrane represented golden standard for systematic reviewsSite is too difficult, would go elsewhereAccessibilityNot evaluatedFindabilityFinding the websiteFinding the site was an obstacle for the majority of participants in Test 1. Despite the fact that 11 of 13 of these participants said they had visited The Cochrane Library before, the same number were not able to find the site without considerable confusion, and six of these 11 did not find the site at all until they were helped by the test facilitator. Although most participants in Test 2 had more success, finding the site remained a problem for some. One of these, a EBM-skilled UK participant, used 23 minutes to arrive at The Cochrane Library from a blank browser page.Much of this trouble stemmed from the participants' failed attempts to find Cochrane through Google search technology. These searches often failed because Google did not rank The Cochrane Library on the top of the first results page when queried for \"Cochrane\" or \"Cochrane Library\". In part this may be due to the fact that only the top few pages of The Cochrane Library were open to indexing in Google, affecting the ranking of the site. Several participants followed other links that appeared higher up on the results list, including links leading to the previous publisher of the site and to The Cochrane Collaboration site, expecting they would lead to the Library. After arriving at these other sites, participants continued to express confusion as to where they were because they found Cochrane-related content.Problems searching for contentFinding specific content was also a major problem once participants arrived at The Cochrane Library. Participants attempted to solve most tasks by performing a search. Even when participants were asked to \"take a look around the site\", 75% started this task with a search. Few of our participants used the advanced search functionality. The simple search was the single most used feature in these tests, and many of these searches failed, leaving participants with a negative impression of the search functionality in the Library. Some participants compared The Cochrane Library to PubMed search, which they found easier to use.Misspelling was the most common search-related mistake made by non-English participants. They were used to getting help with this from other search engines that was not provided by The Cochrane Library search: \"If I get the spelling wrong, Google will help\". Another problem this group experienced was recalling precise terms (for instance recalling \"overweight\" but not \"obesity\"). The publisher redesigned parts of the search interface after Test 1. However in Test 2 the non-native English participants still had considerable problems finding content, mainly due to problems with spelling and recall of correct terms.Search results were often misinterpreted. One of the most critical problems we observed was participants' confusion regarding what they were finding. Many participants did not notice that hits occurred in different databases in The Cochrane Library and thought all hits were completed Cochrane Reviews. We observed participants clicking on and reading review protocols and reports of individual clinical trials, mistaking them for systematic reviews.The search engine was also too sensitive. For instance \"huntingtons\" gave no hits, while \"huntington's\" did. \"Keywords\" option did not provide stabile results.Participants were also confused when their searches produced few or no search results. Some misinterpreted getting few hits as being the result of a bad search. The concept underlying the Cochrane Database of Systematic Reviews of one review per subject did not seem apparent. In addition, non-native English speakers interpreted a lack of hits as a result of their own bad English even though this might not have been the case.Problems browsing for contentTest persons did not browse much, though this may have had to do with their problems understanding the organisation of the site. Few people were able to describe how the content was structured by viewing the front page and nobody could point to a menu with any certainty. Only one test person used the \"Topics\" entry at the top of the front page, though it was not apparent whether other participants did not see it or preferred not to use it.UsabilityLanguage and jargonParticipants reacted to the use of jargon throughout the site. Some of the jargon was site-specific (such as the term \"record\" which led to full texts) and some was tied to research terminology (for instance \"protocol\"). The use of jargon gave the impression that the site was for academic use only and effectively discouraged participants from using several of the site's functions.Legibility and layoutMost felt that there was too much text on the front page and that the type was too small. The participants that clicked on the \"Help\" and the \"More Information\" section also found them very dense.\"It's very messy. Do I have to read all of this?\"There was lots of frustration about the screen being taken up by other things than the review text such as the top banner space. Several participants made negative comments about having to scroll down to see full front page.\"The actual content is stuck in this little area down here.\"Reading patternWe were interested in how participants read reviews and asked them to show us how they normally would approach document if they had limited time (two to five minutes). Most referred to the conclusion section. Several said they would read the abstract, while some mentioned the objectives, results, and background sections. Most said that they normally would not be interested in reading a whole review.We asked participants specifically about the forest plot graphs in the Cochrane Reviews, as they present a lot of information in a summarized form that could be useful for a reader in a hurry. Some participants found them helpful; others found them confusing. They were very difficult to comprehend for those participants who had not seen them before, and were not intuitively located.CredibilityWhen asked if they would trust the information on The Cochrane Library, all participants replied that they would, often because of a familiarity with the Cochrane name and more or less vague ideas about the quality of Cochrane products: \"because it's very respected\"; \"it's a reputable name\"; \"because I've heard good things about it.\"In Test 1, however, we observed potential challenges to this trust because of confusion about site/content ownership. This was primarily tied to the prominence of the Library's publisher Wiley-Blackwell on the website. Wiley's logo was placed higher up on the page than Cochrane's, and Wiley's Home, About Us, Contact Us, and Help buttons were assumed to be Cochrane Library buttons by most participants. Participants who used these buttons often did not realise that they were no longer in The Cochrane Library. When asked to describe the relationship between Wiley and The Cochrane Library, many described The Cochrane Library as a sub-group of Wiley:\"It gives me sort of pharmaceutical industry associations. I think that The Cochrane Library is a subgroup (of Wiley).\"Several changes were made to the website in order to address these issues after Test 1, and participants in Test 2 did not display the same confusion.We also observed that The Cochrane Library's perceived credibility could be over-interpreted. The only contents on The Cochrane Library that are \"Cochrane approved\" are the reviews listed in the Cochrane Database of Systematic Reviews. Despite this fact, some participants assumed that everything in the Library was \"Cochrane-approved\", including the trials, reviews and reports in the individual databases: \"This will just have things that Cochrane have looked at\"; \"If I was looking for a piece of evidence and I found it on Cochrane I would think that it was high quality.\"UsefulnessSome participants assumed that The Cochrane Library only dealt with medical topics and did not expect to find information on topics such as dentistry, nutrition, or acupuncture. The Library was also perceived by some as primarily an academic resource: \"I've tended to think that this is where researchers go to add to the body of knowledge or to see what there is, they'd use this (to build up) Clinical Evidence or Bandolier.... but if I was wanting to get back to the source of information, this is where I would want to go.\"The website has attempted to signal that it is a resource for all types of healthcare decision-makers by adding buttons on the front page entitled \"For Clinicians\"; \"For Researchers\"; \"For Patients\"; and \"For Policy makers\". These lead to short descriptions of what The Cochrane Library can offer each of these groups. However, while some participants thought these were advertising because of their position in the right-hand column, several others assumed that they led to specially adapted versions of The Cochrane Library, and were disappointed when this turned out not be the case:\"I'm surprised that there's a link through to patients here. (...) I didn't realise that it was so well-developed along those lines.\"\"Oh, so it's an (advert)... I was hoping it would give me a tailored search programme, a bit like NLH, which asks you \"are you a GP...\"Others disliked these distinctions between different target groups: \"I don't know why clinicians should differ from researchers. We all need to have \"high quality information at our fingertips.\"Several participants were positive to the fact that patients' information needs were being addressed in the form of the Plain Language Summaries they found in the Cochrane Reviews. They saw these products as helpful both for communicating with patients and for understanding the research results themselves.\"I wouldn't want to go and read all the nitty gritty. The short bits, the one page was useful.\"DesirabilityTwo thirds of the participants complained that the site looked messy and difficult to use, that there was too much information. All expressed frustration with failed attempts to find relevant content. Participants wanted a web site they could get into quickly, find what they were looking for, and get out again. \"Crowded,\" \"busy,\" \"cluttered,\" \"a lot going on,\" \"difficult to find any one particular thing\" were typical comments. Some participants felt \"overwhelmed,\" \"bombarded\" and \"stupid.\"While most expressed interest in this type of evidence-based resource, many were cautious, or concerned that they lack the necessary skills: A nurse commented: \"This is maybe more for doctors.\" A physician who had trouble finding specific content chose to search for \"dementia\" during a test task, and explained why: \"That's kind of how I'm feeling right now.\"ValueAt the beginning of the test all participants said they expected to be able to find content that was relevant for them on The Cochrane Library. Most felt that Cochrane Reviews represented the golden standard for systematic reviews. Many were put off by the amount of information and concerned about the time it would take them to find what they were looking for.\"Not easy to get around\"; \"Most of us don't have time to get around\"; \"So many pages are better designed, so you just get fed up and frustrated and go somewhere else.\"DiscussionOur study shows that health professionals' experiences of The Cochrane Library were considerably less than optimal. Test participants had much difficulty locating both the site and the evidence. Non-native English speakers were at an extra disadvantage when retrieving relevant documents. Many participants displayed feelings of ineptitude, alienation and frustration. Some made serious mistakes in correctly identifying different information types. Although nearly all expressed a high regard for the credibility of The Cochrane Collaboration, some later displayed a mistrust of the independence of the information. Others were overconfident, thinking everything on The Cochrane Library site had been quality-approved through an editorial evaluation, transferring the quality association they had of Cochrane Reviews to the entire content of the library.There are few published usability studies of health professionals using online health libraries or other similar collections of evidence-based medical literature. A commercial company carried out parallel testing of The Cochrane Library for Wiley-Blackwell in 2005 and 2006. Their unpublished reports showed findings that were by and large similar to ours, though included only participants living and working in the UK and therefore did not duplicate the problems we found regarding non-native English speakers. One usability study of an NHS website published in 2003 [17] found that major problems were often caused by specialized library terminology. This supports our findings regarding unfamiliar language and jargon. The few other usability studies of health-related web sites we uncovered dealt with online information for patients or the public.Our results were used in discussions with The Cochrane Collaboration Steering Group and the publisher, Wiley-Blackwell, in order to develop and improve The Library web site. Other publishers of evidence-based content could use the more generic results to improve their own websites.Searching (and finding): critical to evidence-based practiceThe Cochrane Library site is not alone in having problems with findability. Results from usability tests of 217 web sites performed by Jakob Nielsen's team showed that search functionality and findability are the two largest categories of usability problems leading to task failure [7]. However, it is particularly ironic that a website built specifically to support evidence-based health care by synthesizing, organising and making accessible an overwhelming amount of health research should itself be perceived as overwhelming and difficult to navigate.Discriminating designIn this study the non-native English speakers, though displaying no visible trouble reading English text, were at an extra disadvantage when trying to search. Their problems were related primarily to difficulty recalling and spelling query terms that resulted in relevant hits. Creating a reliable base of evidence is a task no organisation or country can solve alone – cross-national efforts are needed. Easy access to a body of high quality evidence should not be limited to native English-speaking participants. There is a wealth of technology that could be used to improve the user experience of searching for non-native English speakers. Spelling aids or query translation from other languages would be particularly helpful to these kinds of users. Automatic query expansion with synonyms (used by PubMed) could provide a better experience both for all searchers but would be particularly helpful for those with a limited English vocabulary.Challenge – building a good mental model for evidence searchingOur findings revealed other challenges for designing good search functionality. In the Cochrane Database of Systematic Reviews, a precise query will result in only one or a few hits, as the underlying concept is one review per topic. However our participants' mental models of how search should function were based both on Google and PubMed, where simple queries produce a great number of results. The concept of a narrow search resulting only in a few hits is clearly still novel to many users and ways in which this can be made clearer need to be explored.Challenge – building a good mental model of evidence-based information hierarchyOur findings showed that systematic reviews can be confused with protocols and reports of clinical trials, even among experienced users who have a clear idea of the difference between these document types. This kind of misinterpretation may happen especially when different document types are mixed together in search results lists. Different document types need to be distinguished from each other, both physically and visually – protocols should possibly be moved to a separate list. The importance of large clear labelling at the top of the individual documents enabling readers to easily distinguish between protocols, reviews and individual studies should also not be underestimated.Appraising the source instead of the documentA related problem is the tendency for users to assume all Cochrane Library contents are Cochrane-approved. Most of our test persons seemed inclined to be satisfied with a quality assessment short-cut: making judgements about the trustworthiness of the publishing source rather than critically assessing individual documents of research as EBM teaching encourages. This inclination, when coupled with poor signposting on a site containing information of varying levels of editorial evaluation and research quality, leaves a gap wide-open for serious misunderstandings about the strength and quality of different pieces of evidence. Blind trust of a whole source is a complex labelling and branding problem and needs to be addressed by publishers on many levels.Fragile credibilityThough Cochrane clearly enjoyed a high reputation among our participants, our study showed that even very small details can cause otherwise trusting users to suddenly question ownership and thereby credibility, such as an \"About us\" button leading to a page with a publishers' (unfamiliar) logo. While a large study from the Stanford Credibility project showed that consumers placed a lot of emphasis on the look of a site [18], a smaller parallel study showed that expert users tended to emphasize the reputation of the source when evaluating the trustworthiness of information found online [19]. Additionally it is important to follow the EBM principles of transparency and make it absolutely clear who is behind information that claims to be neutral and evidence-based.This site is not for someone like me...Many of our participants felt that The Cochrane Library site was for \"researchers\" or others with more knowledge than themselves, in part due to use of unfamiliar or academic jargon, but also connected to their failure to find relevant information. The feelings of ineptitude expressed by participants in this study is perhaps mirrored in the Australian study, where 41% of the participating physicians blamed their own limited search skills as impediments to making better use of research data. In fact, many of the problems our participants encountered were not due to their own lack of skills, but to design flaws that could be solved following usability heuristics [20] and research-based guidelines for web design [7,13,21] or implementing better search technology. It is also important to signal inclusiveness and relevance to other health care areas than just medicine. Clear signs of content produced for patient target groups could also serve to lower the perceived threshold for professionals.Is valuable content enough?Repeatedly we heard praise for the quality of content of this site. But frustration levels were very high, and several participants said they were ultimately too lazy to bother to use a site that made it so difficult for them. Information foraging theory describes user behaviour on the Internet as similar to wild animal's search for food: we want maximum benefit for a minimum of effort [22]. Jakob Nielsen points out that with the development of good search engines, it has become easier for information gatherers to move quickly between different hunting grounds, claiming that web sites should be designed less like big meals and more like tasty snacks, quick both to find and to eat [23]. A resource like Cochrane may be theoretically a great meal for a hungry animal, but too difficult to find and catch to be worth the effort, especially when less challenging prey is more easily available.Limitations of this studyOur goal is to identify the emerging issues rather than to quantify them. In reporting results, we have therefore not emphasized frequencies of events. As our data set has not been designed to statistically represent a set of respondents, presenting numbers can be misleading [24].The user tests were performed in a laboratory setting, and may not reflect actual behaviour or reactions from real-life situations. For instance, increased time pressure in clinical situations may result in even higher degrees of user frustration when an interface does not easily or intuitively produce quick results.UK-based tests were held in the office of The Cochrane Collaboration, and this may have influenced the answers of participants regarding use and attitudes towards The Cochrane Library and Cochrane Reviews, despite our assurances that we were not connected with the design of the web site. Answers regarding familiarity and use of research were self-reported and not empirically validated.The honeycomb model was not used to design the interview questions, only applied in retrospect to our data analysis. This may have affected the relevance of the data we collected to this model. On the other hand, this may have led to less \"leading\" questioning on our part.The Cochrane Library, like most websites, is under continuous development/change, and several of the weaknesses we identified have since been improved.ConclusionRecommendations based on findingsBuilding web sites for evidence-based practice is not much different than building for good web usability in general. However, the consequences of not finding information or of finding the wrong information have potentially critical consequences. Health professionals' user experience of evidence-based online resources can be improved by applying the following principles:- Follow existing usability heuristics and web usability guidelines, designing especially for findability through search engines, as well as for speed of use particularly important to health professionals.- If resources are limited, focus on improving simple (non-advanced) search functionality, including technology that will help non-native English speakers.- Drop \"researcher\" language and jargon to encourage use by health professionals.- Don't assume users possess good mental models of evidence hierarchies. Make document types evident where possible – through information architecture, labelling, and search results design.- Clearly mark the difference between quality-approved content and not quality-approved content.- Ownership and authoring must be clear at all levels of the site for supporting and maintaining credibility.Competing interestsCG is director of the Norwegian branch of the Nordic Cochrane Centre. JC is Co-ordinator & Managing Editor of The Cochrane Anaesthesia Review Group. CG and SR are involved in projects to improve summaries included in Cochrane Reviews. Test 2 was carried out in collaboration with Wiley-Blackwell, and partly funded by them.Authors' contributionsSR conceived of and designed the study, carried out user testing, data analysis and interpretation, and drafting of the manuscript. CG helped design the study, carried out user testing, data analysis and interpretation, and drafting of the manuscript. JC recruited UK participants, carried out user testing, transcribed and coded the British tests, and commented on the manuscript. All authors read and approved the final manuscript.Pre-publication historyThe pre-publication history for this paper can be accessed here:Supplementary MaterialAdditional file 1Appendix. Interview guide used in Test 2.Click here for file\n\nREFERENCES:\nNo References"
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+{
+    "id": "PMC2529287",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2529287\nAUTHORS: Antoine Parrot, Martine Antoine, Antoine Khalil, Jonathan Théodore, Gilles Mangiapan, Bernard Bazelly, Muriel Fartoukh\n\nABSTRACT:\nBackgroundThere are limited series concerning Dieulafoy disease of the bronchus. We describe the clinical presentation of a series of 7 patients diagnosed with Dieulafoy disease of the bronchus and provide information about the pathological diagnosis approach.Patients and methodsA retrospective review of patients who underwent surgery for massive and unexplained recurrent hemoptysis in a referral center during a 11-year period.ResultsSeven heavy smoker (49 pack years) patients (5 males) mean aged 54 years experienced a massive hemoptysis (350–1000 ml) unrelated to a known lung disease and frequently recurrent. Bronchial contrast extravasation was observed in 3 patients, combining both CT scan and bronchial arteriography. Efficacy of bronchial artery embolization was achieved in 40% of cases before surgery. Pathological examination demonstrated a minute defect in 3 cases and a large and dysplasic superficial bronchial artery in the submucosa in all cases.ConclusionDieulafoy disease should be suspected in patients with massive and unexplained episodes of recurrent hemoptysis, in order to avoid hazardous endoscopic biopsies and to alert the pathologist if surgery is performed.\n\nBODY:\nBackgroundMassive hemoptysis is a life threatening condition associated with a mortality rate exceeding 50% in the absence of adequate treatment [1-3]. A standardized and prompt management is required in emergency to improve survival, as well as a careful search of both the location and the etiology of bleeding. More than one hundred causes of massive hemoptysis have been described [4]. Nevertheless, no cause is identified in about 15% of the cases, despite a complete investigation including fiberoptic bronchoscopy and CT-scan [5,6].We have recently reported a few subset of patients with so-called cryptogenic hemoptysis, in whom an unexpected vascular abnormality was demonstrated at pathological examination of the pulmonary resection, characterized by a bronchial artery running within the submucosa and called Dieulafoy disease of the bronchus [7]. Additionally, only eight cases of bronchial Dieulafoy disease proved on histological data in six case reports have been reported to date [8-13]. To our knowledge, no case series have been published from a tertiary referral center managing massive hemoptysis.The aim of our study was to report the clinical presentation of the patients who underwent surgery for massive hemoptysis in our center and in whom a Dieulafoy disease of the bronchus was eventually diagnosed and to provide detailed information on the approach to pathological diagnosis in this field.Patients and methodsStudy designAll the patients were recruited in a respiratory intensive care unit of a 800-bed tertiary university hospital in Paris, France, between May 1995 and July 2006. During the study period, 810 patients were admitted to our unit for hemoptysis, 111 of whom underwent surgery. Part of those patients has already been reported from our group [7,14]. The study was conducted according to the French law which judged unnecessary ethical approval and patient consent for such a retrospective analysis of medical records.Diagnosis of Dieulafoy disease of the bronchusThe diagnosis of Dieulafoy disease of the bronchus was clinically suspected in case of the need for surgery for massive hemoptysis with no identifiable cause after fiberoptic bronchoscopy and CT-scan. Dieulafoy disease of the bronchus was pathologically confirmed by the evidence of an isolated and localized area of hemorrhage with no underlying lung disease known to be associated with bronchial systemic hypervascularization (such bronchiectasis) on macroscopic inspection plus the evidence of an unusual large superficial bronchial artery located into the submucosa, possibly extended through the mucosa into the bronchus lumen with no vasculitis, aneurysm or arteriosclerosis on microscopic examination.Pathologic lung studyThe lung samples (lobectomy) were processed in an uniform way by the same pathologist (MA), when Dieulafoy disease was suspected. First, the fresh lung resection was macroscopically examined. Macroscopic inspection confirmed the focal nature of the hemorrhage. Nodular lesions, blebs, areas of induration and subpleural hypervascularization were checked. Segmental and sub segmental bronchi were subsequently opened with chisel in the absence of obvious etiology. The presence and the location of blood cloths were identified. After careful washing, bronchiectasis, bronchial inflammatory aspect and tumoral obstruction were searched as well as mucosal abnormalities, i.e. minute defect and ulceration. Second, after formalin fixation, 3 mm thick serial sections were performed perpendicularly to the axis of the suspected bronchus previously identified by the presence of cloths, focal areas of hemorrhage or mucosal abnormalities; the same procedure was applied to the other bronchi for comparison. Samples of fixed tissue were processed into paraffin block, perpendicularly to the bronchial axes. Third, serial sections were performed on the paraffin block focusing on the suspected bronchus until lesions were found. Sections were stained with hematoxylin-eosin-safran, and elastic stain (Miller stain) for vessel identification.Collection of patients' dataThe following prospectively collected clinical data were extracted from our data base and controlled with the review of the medical charts: baseline demographics, drug intake, comorbid conditions, severity of hemoptysis, clinical presentation, laboratory tests, chest radiography, fiberoptic bronchoscopy and CT scan, ICU management and vital status at ICU discharge.ResultsSeven patients fulfilled the aforementioned criteria during the 11-year study period.Patients' characteristicsThe patients (5 males) were aged 54.3 ± 11.5 years (range, 38 to 69 years). They were current heavy smokers (49 ± 28.5 packs/years); all but one were alcohol abusers. Two had mild to moderate chronic obstructive pulmonary disease. One patient had a history of pulmonary tuberculosis. Two patients were treated for systemic hypertension, 1 of whom had a history of a transient ischemic stroke and unexplained intestinal bleeding (Additional file 1). A previous episode of hemoptysis had occurred in 5 patients, 3 of whom had previously received a bronchial artery embolization (BAE). Of note, 1 patient (patient n°4) had been managed in our center 19 months ago for a first episode of massive hemoptysis (amount of 400 ml). Four fiberoptic bronchoscopies were necessary to locate the bleeding in the left upper lobe, as CT scan showed bilateral ground glasses. A successful BAE was performed. The patient refused a secondary scheduled surgery despite the staff decision.The cumulative amount of bleeding ranged from 350 ml to more than 1000 ml on admission to our unit. There were, however, mild clinical and biological consequences of the bleeding. Bedside chest X-ray was unremarkable. High resolution (n = 5) or multidetector (n = 2) CT-scan angiography showed ground glass opacities in all patients, that were isolated or associated with alveolar opacities (n = 4). The upper lobes were mainly involved (n = 5). Of note, a frank contrast media extravasation within the lumen of the bronchus related to the focal hemorrhagic area was evidenced using multidetector CT-scan angiography (patient n°4). There was no lung parenchyma abnormality suggestive of carcinoma, bronchiectasis or tuberculosis. A flexible fiberoptic bronchoscopy was performed within the first 24 hours of admission, to locate both the side and site of the bleeding. Overall, 10 bronchoscopic procedures were performed. A bilateral bronchial flooding by blood was evidenced in all patients. The location of the bleeding was successfully performed after bronchoscopic techniques in 5 patients.ManagementThe therapeutic management was standardized, as described elsewhere [14]. Bronchoscopic techniques were attempted to control the bleeding, combining blood aspiration and local instillation of cold saline lavage. Vasoconstrictive agents were delivered bronchoscopically (adrenalin, n = 4), intravenously (terlipressin, n = 1) or both (n = 1). A BAE was first attempted in all patients totaling 10 sessions before surgery (Figure 1). Technical failure of bronchial arteriography was related to failure of canulation in 2 patients (patients n°1 & n°3) and anatomical consideration in 2 others (patients n°5 & n°6). All bronchial arteries draining the bleeding site were enlarged without systemic to pulmonary artery shunting. A frank contrast media extravasation into the bronchial lumen was evidenced in 2 patients (patients n°2 & n°7). Altogether, BAE was completed in 5 cases (4 patients) and controlled the bleeding in 2 cases (2 patients). According to the high initial amounts of bleeding, all the patients were secondary referred for surgery after 6.7 ± 5.8 days (median time 5 days). The lobe to remove, from which the bleeding originated, was identified by the combination of clinical examination, chest X-ray, CT scan and bronchoscopic findings. Bronchial arteriography was not used for locating the bleeding.Figure 1Bronchial arteriography efficacy. ASA = anterior spinal artery; RBICT = right broncho-intercostal trunk.Follow-up after surgeryAll patients were alive at hospital discharge. Follow-up data were available for a mean duration of 35 ± 30 months (range, 6 to 96 months) after surgery. All but one patient (patient n°4) remained free of bleeding recurrence during follow-up. This latter patient was treated successfully with BAE. The patient n°7 died from an ischemic stroke 12 months later.Pathological findingsLung macroscopic examination located the pathological area in all patients by showing a focal hemorrhagic area within the parenchyma and identifying a localized clot in the corresponding segmental (n = 5) or sub segmental bronchus (n = 2) (Additional file 2). Moreover, a minute bronchial mucosal defect was eventually observed (n = 3) (Figure 2A). There was no evidence of bronchial or vascular chronic disease. The macroscopic inspection was unremarkable, except blebs (patients n°1 & n°2) and nodular mass (patient n°6) that were distant from the pathological hemorrhagic area.Figure 2Pathological Findings. Macroscopic view showing a minute defect within the bronchial tree (arrow) (A). Low-power view showing the location of the vessel in the sub-mucosa beneath the cartilage plate, and the presence of the material of embolization in the lumen (arrow) (B). High-power view revealing the protrusion of the superficial vessel in the lumen with an ulceration and a squamous metaplasia of the epithelium (C). High power-view showing a dysplastic artery with elastic stain (Miller stain) (D).Microscopic examination revealed a large and dysplasic superficial bronchial artery in the submucosa in all patients beneath the cartilage (Figure 2B). Artery ulceration with rupture into the bronchial lumen was observed in 3 patients (Figure 2C). The structure of the bronchial artery appeared dysplastic with irregular thickness of the wall, which was either fibrotic or rich in elastic fibrils and had a tortuous appearance when cut at various angles. These arteries appeared on Miller stain with a thick internal lamina and a thin external one, in favor of the bronchial nature of the artery (Figure 2D). Finally, we observed (patient n°4) the material of embolization in the lumen of the vessel in 1 patient (Figure 2B). The respiratory epithelium was either clearly or slightly eroded and appeared sometimes metaplastic, although the structure of the bronchus remained normal. Of note, the nodular mass aforementioned (patient n°6) was related to a granulomatous disease consistent with an old sarcoidosis. In another patient (patient n°1) a deposit of amyloidal structure was identified. Otherwise, histological examination was unremarkable. No evidence of chronic bronchial or other vascular disease was identified. No microorganisms were grown, after special staining and culture for mycobacteria and fungi.DiscussionOur study aimed at better describing the process for diagnosing the Dieulafoy disease of the bronchus, from the clinical suspicion to the pathological confirmation, based on a series of 7 patients who underwent surgery for massive hemoptysis in a referral center over a 11-year period. The condition was clinically suspected in heavy smokers with recurrent and unexplained episodes of massive hemoptysis, characterized by amounts of bleeding both high and rather disproportioned, as compared with those usually reported in patients presumed to have a cryptogenic hemoptysis. Although there were no specific CT-scan or angiographic criteria, the frequent findings of both the direct and frank contrast media extravasation within the suspected bronchial lumen and the enlarged aspect of the bronchial artery without systemic to pulmonary artery shunting were suggestive of the vascular anomaly. A subsequent structured and rigorous pathological examination of the surgical lung resection confirmed definitively the diagnosis.Dieulafoy disease is a vascular anomaly characterized by the presence of a dysplastic artery in the submucosa. It was first reported in the gastrointestinal tract, accounting for up to 2% of the bleedings [15]. The disease has been recently described in the respiratory tract [12]. However, the incidence of the Dieulafoy disease of the bronchus is unknown and probably slightly underestimated regarding to the rigorous pathological procedure needed for establishing the diagnosis. In our experience, Dieulafoy disease of the bronchus accounted for at least 6% of the patients undergoing surgery for hemoptysis overall and up to 55% of the patients undergoing surgery for hemoptysis presumed to be cryptogenic [7].The diagnosis of Dieulafoy disease of the bronchus should be suspected on the combination of history, clinical features and imaging investigations in order to consider surgical treatment and alert the lung pathologist. First, our patients were heavy smokers, similarly to the previous published isolated case reports. Second, respiratory past history was unremarkable, except unexplained and severe episodes of hemoptysis in 5 patients, 3 of whom had received a BAE (Additional file 1). Third, hemoptysis was massive and presumed to be cryptogenic, since no cause was identified after physical examination, fiberoptic bronchoscopy and CT-scan. Conversely to the gastrointestinal disease for which the endoscopic findings are diagnostic, we did not use bronchoscopic criteria to diagnose the vascular disease because the source of bleeding may be difficult to assess during active massive hemoptysis [16-18], the bronchial abnormalities may be sub segmental and therefore not accessible and the small size of the bronchial lesion (usually less than 10 mm) may be difficult to detect when surrounded by clots. However, a few mucosal abnormalities have been bronchoscopically described in this setting, such as a smooth elevated non pulsating lesion [13] or a nodular lesion within a normal overlying mucosa [9]. It should be emphasized that these later bronchoscopic findings are not specific and may be related to bronchial artery aneurysms, arteriovenous malformations or small cancers [19-21]. As the amount of bleeding related to Dieulafoy disease may be massive, bronchial biopsies should be avoided in this setting, even during a period of non active bleeding [9,13]. Moreover, in our opinion, performing biopsy should be not useful in this setting, since the diagnosis of Dieulafoy disease of the bronchus should be based on the pathological examination of a large surgical lung resection. Fourth, the bronchial arteriography findings did neither evidence systemic to pulmonary shunts nor aneurysms. Additionally, the arteries appeared all enlarged and frank contrast media extravasation in the bronchial lumen was frequent, when combining the findings of both BAE (n = 2) and multidetector CT-scan angiography (n = 1). These findings are in accordance with those reported by Durham et al in gastrointestinal Dieulafoy disease [22]. Conversely, little angiographic data are available regarding to the bronchial artery disease [8-10,12,23,24]. Dilated vessels have been described [9,10,23], especially in association with specific parenchymal diseases [10,23]. Last, although no firm conclusions can be drawn regarding to the small size of our population, our study highlights the poor efficacy of BAE in this clinical setting, as compared with the usual 80% to 90% successful rate of bleeding control using this procedure [17]. Nevertheless, owing to the morbidity and the mortality related to emergency surgery performed during active bleeding, we recommend to attempt BAE as the first-line therapeutic approach [7]. Additionally, some patients with a non-diagnosed Dieulafoy disease may have probably been treated with BAE.To our knowledge, our series is the first to carefully describe the pathological investigation to diagnose the Dieulafoy disease of the bronchus on surgical lung resection. The main pathological criterion is the evidence of a large and superficial bronchial artery located within the sub mucosa [8-13]. In our series, the macroscopic analysis was crucial to detect a minute mucosal defect, as usually observed in the Dieulafoy disease of the gastrointestinal tract. As ectopic bronchial arteries have also been described during chronic pulmonary diseases [17,25,26], a special attention was made to exclude chronic lung diseases, such bronchiectasis and other inflammatory processes or carcinoma. Additionally, CT scan demonstrated no parenchymal abnormalities, except ground glass or alveolar opacities reflecting the severity of bleeding [27]. Although a few parenchymal abnormalities (blebs, nodular mass and amyloidal deposit) were pathologically evidenced, those later were actually distant from the focal hemorrhagic area and consequently not considered as the cause of bleeding.The pathogenesis of Dieulafoy disease remains unclear. Whether the origin of the anomaly is congenital, acquired or merely a variation of normal is not known. As suggested in our study, age and/or tobacco use may be predisposing states to the occurrence of the disease. Even if the dysplastic artery wall change may contribute to its weakness, the trigger factor of the vessel rupture is not known. Does the vessel rupture occur after a mucosal injury or does the vessel pressure induce a mucosal defect is unclear. Furthermore, the nature of the vessel that is bleeding remains controversial. Some have suggested that the artery belongs to the pulmonary vasculature owing to the failure of BAE, while others identified the vessel as originating from the systemic vasculature [10,13]. In our series, the angiographic data are supporting this latter hypothesis, as well as the pathological findings of the vessel with elastic stain and the intravascular evidence of material of embolization (patient n°4) (Figure 3B). Last, Dieulafoy disease might be a part of a general disease as suggested by the clinical history and outcome of the patient n°4.The limitations of our study are related to its retrospective nature and to the fact that it was conducted on patients undergoing surgery in a referral center with an extensive experience of severe hemoptysis.In summary, Dieulafoy disease of the bronchus should be considered in a heavy smoker patient with unexplained and recurrent massive hemoptysis, after a rigorous confrontation of both clinical and radiological findings. Useless and dangerous bronchial biopsies should be avoided. The therapeutic approach should be surgical and the pathological examination should be structured and meticulous.AbbreviationsBAE: Bronchial artery embolization.Competing interestsThe authors declare that they have no competing interests.Financial supportNoneAuthors' contributionsAP had full access to the data and takes responsibility for the integrity of the data at the accuracy of the data analysis. MA performed the pathological analysis. All authors read and approved the final manuscript.Supplementary MaterialAdditional file 1Table S1. Patients' characteristics, management and outcome. Comparison with the literature cases.Click here for fileAdditional file 2Table 2. Pathological findings. Comparison with the literature cases.Click here for file\n\nREFERENCES:\nNo References"
+}

batch_11/PMC2529288.json

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+{
+    "id": "PMC2529288",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2529288\nAUTHORS: Anita L Sullivan, Timothy Dafforn, Pieter S Hiemstra, Robert A Stockley\n\nABSTRACT:\nBackgroundSecretory leukoproteinase inhibitor (SLPI) is an important inhibitor of neutrophil elastase (NE), a proteinase implicated in the pathogenesis of lung diseases such as COPD. SLPI also has antimicrobial and anti-inflammatory properties, but the concentration of SLPI in lung secretions in COPD varies inversely with infection and the concentration of NE. A fall in SLPI concentration is also seen in culture supernatants of respiratory cells exposed to NE, for unknown reasons. We investigated the hypothesis that SLPI complexed with NE associates with cell membranes in vitro.MethodsRespiratory epithelial cells were cultured in the presence of SLPI, varying doses of proteinases over time, and in different experimental conditions. The likely predicted charge of the complex between SLPI and proteinases was assessed by theoretical molecular modelling.ResultsWe observed a rapid, linear decrease in SLPI concentration in culture supernatants with increasing concentration of NE and cathepsin G, but not with other serine proteinases. The effect of NE was inhibited fully by a synthetic NE inhibitor only when added at the same time as NE. Direct contact between NE and SLPI was required for a fall in SLPI concentration. Passive binding to cell culture plate materials was able to remove a substantial amount of SLPI both with and without NE. Theoretical molecular modelling of the structure of SLPI in complex with various proteinases showed a greater positive charge for the complex with NE and cathepsin G than for other proteinases, such as trypsin and mast cell tryptase, that also bind SLPI but without reducing its concentration.ConclusionThese data suggest that NE-mediated decrease in SLPI is a passive, charge-dependent phenomenon in vitro, which may correlate with changes observed in vivo.\n\nBODY:\nBackgroundSLPI is an efficient inhibitor of NE and other serine proteinases[1,2] and is found in high concentrations in secretions such as respiratory mucus[3]. In vitro it has antibacterial [4-6] and antifungal[7] properties and has been shown to prevent viral infection[8,9]. In addition it has anti-inflammatory properties distinct from inhibition of extracellular NE, that are potentially important in host defence and auto-immune conditions [10-14]. These properties suggest that SLPI may be important in diseases such as bronchiectasis and COPD that are characterized by neutrophilic inflammation and infection. SLPI should be protective in these conditions, and indeed patients with chronic bronchitis (CB) have a higher concentration of SLPI in lung secretions than healthy controls[15], probably because of submucosal gland hypertrophy and increased serous cell secretion. However the amount of SLPI found in the sputum of patients with CB and bronchiectasis decreased during exacerbations [16-18] and increased again in the stable state[16,18]. It was also lower in the stable state in sputum from patients with frequent exacerbations compared to those with infrequent exacerbations [19], suggesting that low concentrations of SLPI increase the risk of developing an exacerbation. In addition it was lowest in those patients with the greatest neutrophilic inflammation in the stable state[20], and lower in those who remained colonized in the stable state compared to those who cleared their infection[18,21]. This relation to infection has been confirmed in studies showing that SLPI was also reduced in vaginal secretions in the presence of bacterial infection [22].In general, SLPI and NE concentration appear to be inversely related in sputum in COPD and CB[23] but the reason for this relationship in vivo is not understood. The genetics of patients with early-onset COPD have been investigated and no mutations, deletions or disease-associated polymorphisms in the SLPI gene have been described[24]. In our studies over 20 years, we have not identified any patients with an absence of SLPI in sputum. Therefore at present the concept that a primary deficiency of SLPI initiates inflammation or infection is not well supported by the available data.A number of studies have described an inverse relationship between SLPI and NE in vitro using tissues ranging from primary nasal and bronchial epithelial cells, through a variety of cell lines, to isolated tracheal submucosal glands [25-28]. Since cell culture supernatant from cells exposed to NE has a lower concentration of SLPI protein than supernatant from cells not exposed to NE, neutrophilic inflammation may directly predispose to low SLPI in vivo. Previous in vitro work also included measurement of SLPI gene expression in cells exposed to NE and high levels were found to increase gene expression [26-29]. The fall in protein must therefore relate to alterations in post-transcriptional events. Immunohistochemical studies of primary nasal epithelial cells showed greater SLPI protein in cells treated with NE than those not treated, suggesting failure of secretion or redistribution of the protein rather than failure of synthesis[28]. Studies of NE-treated cells lysed using a detergent-based solution after removal of media demonstrated that the SLPI not found in cell culture media was in the cell lysate [27].One mechanism that might explain the NE-induced redistribution of SLPI protein hinges on the positive charge of the NE molecule, which may enable it to associate with cell membranes [30]. Once associated with epithelial cell membranes, it could bind SLPI, although not as efficiently as in free solution[31], and would hence remove SLPI from cell supernatant. The known structures of NE[32] and SLPI[33] suggest that charged residues may remain exposed on the outside of each molecule when complexed with each other, so the NE-SLPI complex is likely to have a more potent positive charge and therefore is more likely than SLPI alone to associate with cell membranes and negatively-charged proteins.Other possible mechanisms include failure to secrete, or binding of SLPI to other cell surface proteins exposed by NE. For example, SLPI has been shown to bind specifically to annexin II[34] and to scramblase[35], and these may potentially be upregulated or exposed in the presence of proteinases. It is known that there is a receptor specific for the complex of NE and α1 antitrypsin (α1 AT) [36], and therefore it is also possible that the SLPI-NE complex binds to a specific receptor. Finally, there may be a mechanism to import SLPI actively into cells. Studies with neutrophils[37], megakaryocytes and platelets[38] show SLPI inside cells, and a recent study indicated that macrophages can import exogenous SLPI into both the cytoplasm and nucleus[12].In the present study, we have investigated the hypothesis that charge-related association of the SLPI-NE complex with cell membranes is the primary mechanism reducing SLPI concentration in cell culture supernatants from SLPI producing lung epithelial cells in the presence of NE. We have conducted experiments investigating the nature of the effect of NE on SLPI protein levels in cell culture supernatant, and pursued preliminary studies into the localization of SLPI in these cells when exposed to NE.MethodsProteinases and inhibitorsNE was purified from empyema fluid using the method of Martodam et al[39]. Absence of endotoxin contamination was confirmed with the Limulus amoebocyte assay (E-TOXATE, Sigma, UK). Activity of NE was measured using the synthetic chromogenic substrate N-succinyl-(ala)3-p-nitroanilide (Sigma, UK). Cathepsin G (CG) was similarly obtained from empyema fluid and activity was measured using N-succinyl-phe-pro-phe-p-nitroanilide (Bachem, UK). Porcine pancreatic elastase (PPE) was obtained from Sigma and the activity was measured using N-succinyl-(ala)3-p-nitroanilide. Trypsin was cell culture grade (Invitrogen) and activity was measured using N-benzoyl-L-arginine ethyl ester hydrochloride (BAEE) (Sigma). Human mast cell chymase and tryptase were obtained from Elastin Products Co. and activity was measured using N-succinyl-val-pro-phe-p-nitroanilide and Z-gly-pro-arg-p-nitroanilide respectively (both from Bachem). ZD0892, a small synthetic peptidyl trifluoromethylketone NE inhibitor with an affinity for NE similar to that of SLPI[40], was a gift from Zeneca Pharmaceuticals (Wilmington, USA) and inhibitory function was measured against a known amount of active NE using N-succinyl-(ala)3-p-nitroanilide. Recombinant human SLPI (rhSLPI) was a gift from Amgen and the concentration was confirmed using the SLPI ELISA (see below). Inhibitory activity was determined as for ZD0892.Measurement of SLPI and total proteinSLPI concentration was measured using the R&D Systems ELISA, a sandwich ELISA consisting of a mouse monoclonal antibody for capture and a horseradish peroxidase-conjugated polyclonal antibody for detection. Total protein was measured in microplates using the Bio-Rad assay (Bio-Rad, USA). The ELISA was validated using mixtures of SLPI and NE or CG in varying proportions to ensure that the proteinases did not interfere with quantification of SLPI. Briefly, recombinant human SLPI supplied as standard in the ELISA kit was mixed at varying molar ratios with NE and CG, incubated at 37°C for 30 minutes and the amount of SLPI recoverable was measured using the ELISA.Cell cultureA549 cells, a lung epithelial cell line derived from lung carcinoma, were obtained from the ECACC and cultured in 50/50 F10/DMEM (both from Gibco, UK) with 10% fetal calf serum (FCS) (Gibco). HepG2 cells, a liver epithelial cell line derived from liver carcinoma, were obtained from the ATCC. They were cultured in DMEM containing 10% FCS, antibiotic antimycotic solution, 150 mmol L-glutamine and MEM non-essential amino acids (all from Sigma). Primary human bronchial epithelial cells (PBEC) were obtained from Cambrex (UK) and cultured in the same supplier's recommended basal media with growth supplements according to their instructions. Experiments were carried out with cells between passage 3 and 8, using basal media only.Most experiments were performed on A549 cells and selected experiments were repeated on PBEC or HepG2 cells in standard submerged monolayer culture. All experiments were performed in at least triplicate wells, and results are expressed as mean ± standard error of the mean (SEM) of at least 3 experiments. Cells were cultured to confluence in T75 or T25 flasks (Gibco) and plated out into 12 or 24 well plates (Gibco). Once confluent, the media were changed to serum free media (SFM) (50/50 F10/DMEM for A549 cells, DMEM alone for HepG2 cells and basal media without additives for PBEC). A549 cells and HepG2 cells were cultured in SFM for a further 24 h before experiments were performed. After treatment with the experimental conditions, supernatants were aspirated and stored at -70°C for later analysis for SLPI and protein concentrations. Cells were rinsed with PBS and harvested using trypsin to detach them and viability was assessed where appropriate using trypan blue exclusion. Statistical analysis was performed using SPSS version 12 for Windows.Experiments1. Dose responseA dose response experiment was undertaken to examine the relationship of NE concentration to the reduction of SLPI concentration in cell culture supernatants. The initial concentration of SLPI in the cell supernatants was not known at the outset of an experiment. Preliminary experimental data showed that the typical concentration of SLPI in supernatants of A549 cells after 24 h culture in SFM was between 0.5 and 2 nM (5–23.4 ng/ml). Cells were therefore incubated in SFM alone as control, or SFM containing NE from 0.5 to 5 nM. Initial experiments also included a control consisting of the buffer used to suspend the NE, in SFM at the same concentration as the highest concentration of NE used. Dose response experiments were repeated with CG and using PBEC with both proteinases.2. Effect of other proteinasesProteinases such as PPE have, like NE, been shown to increase SLPI expression[29], although the effect on protein secretion has not been reported. PPE (which does not bind SLPI), human mast cell chymase, trypsin and human mast cell tryptase (which bind SLPI with varying affinity but do not carry a high positive charge) were used to assess whether the degree of binding to SLPI by the proteinase or its ability to associate with cell membranes by charge would modify the ability of the proteinase to reduce SLPI in the supernatant. The effect on SLPI secretion was compared for PPE, trypsin, chymase, tryptase and CG by incubating cells with varying concentrations of each proteinase for 24 h.3. Time courseThe hypothesis of binding of the NE-SLPI complex to cell membranes because of positive charge suggested that addition of NE to conditioned media would cause removal of SLPI that had already been secreted. Confluent PBEC were cultured for 24 h in SFM and at the end of this period an aliquot was removed for baseline analysis and replaced with either SFM or NE at a concentration sufficient to achieve 10 nM in each well. After gentle mixing of the contents of the wells once with a 1 ml pipette, cells were incubated for a further 24 h and aliquots of 25 μl were taken at various time points within this period and stored at -70°C for analysis by ELISA. Similar experiments were repeated on A549 cells using both NE and CG, and with 1 nM rhSLPI in DMEM added to HepG2 cells followed by NE or SFM control. HepG2 cells were used as a control cell line that does not express or secrete SLPI, to demonstrate the effect of proteinases on SLPI concentration without any possibility of secreted SLPI contaminating the results.4. Effect of a synthetic NE inhibitorAddition of NE inhibitors to culture media containing NE has been shown to abrogate the fall in SLPI protein[27]. If binding of NE to SLPI is important in the mechanism of the fall in SLPI protein, then the timing of the addition of the inhibitor will be equally important. NE was incubated for 30 min at 37°C with a tenfold excess of ZD0892. The mixture of NE and ZD0892 was added to the supernatant of A549 cells that had been serum-starved for 24 h, to achieve a final concentration of 10 nM NE and 100 nM ZD0892. This was compared with NE 10 nM added 10 minutes prior to the addition of 100 nM ZD0892, and ZD0892 100 nM added 10 minutes prior to the addition of NE 10 nM. Control cells were treated with NE 10 nM alone, ZD0892 100 nM alone, or SFM alone. All mixtures were incubated for a further 10 minutes and the supernatants were then aspirated and stored at -70°C for analysis by ELISA.5. Localization of SLPI in cell culture systemGiven that many proteins associate non-specifically with charged surfaces such as cell culture plastic, we speculated that the NE-SLPI complex may associate more with cell culture materials than SLPI alone. This was tested by comparing the concentration of SLPI in solutions added to empty tissue culture plates with the concentration of SLPI in solutions added to wells containing confluent cells, with and without NE. SLPI was added to SFM to achieve a concentration of 1 nM, and the solution was incubated in 12 well plates for 10 minutes, a baseline aliquot was taken for measurement of SLPI and replaced with SFM or NE to achieve a final enzyme concentration of 10 nM. After a further period of incubation the contents of the wells were harvested for measurement of SLPI concentration. In order to block the effect of non-specific binding, both 12 well plates containing cells and 12 well plates without cells were also incubated with media containing 1% w/v human serum albumin (HSA) and SLPI at 1 nM. The effect of 10 nM NE was assessed as before in both sets of plates. Plates were also coated with HSA prior to addition of cells, and the above experiments were repeated using plates with or without A549 cells. Finally in cell-free conditions, the effect of 1% Tween 20 (polyoxyethylene sorbitan monolaurate, Sigma) was assessed in the same way.6. Two compartment modelSLPI is secreted both basally and apically by cells in vitro[41], and in vivo epithelial and glandular cells might be exposed to NE release from neutrophils at both sites during neutrophil migration. To examine whether this may influence the secretion of SLPI, a two compartment model was studied. Transwells (Transwell PET 0.4 μm pore size 12 mm inserts, Corning Life Sciences) were coated with human placental collagen (Sigma) at 50 μg/ml and dried in air. A549 cells were added to the upper compartment in 500 μl media, with 1.5 ml media in the lower compartment. As soon as the cells appeared as a confluent monolayer, media were replaced by SFM at the same volumes. After a further 24 h incubation, reagents were added to upper and lower compartments. Triplicate wells received either SFM alone in both compartments or NE 10 nM in one compartment with SFM in the other. Supernatants were aspirated after a further 10 minutes incubation, and stored at -70°C for later analysis for total protein and SLPI concentration.7. Theoretical modelling of the external charge of the complex between SLPI and proteinasesThe modelling procedure involved the use of the X-ray crystal structure of SLPI with chymotrypsin (structure file kindly provided by Professor Wolfram Bode as described previously[33]) as a template to construct analogous complexes with other proteinases. X-ray crystal structures of NE (PDB ID 1H1B; [42]), CG(PDB ID 1T32;[43]), trypsin (PDB ID 1TX6;[44]), mast cell chymase (PDB ID 1T31;[43]) and mast cell tryptase (PDB ID 2BM2;[45]) were obtained from the Research Collaboratory for Structural Bioinformatics Protein Data Bank[46]. These structures were then superimposed on the proteinase in the SLPI-proteinase complex using SWISSPDB[47]. The values for the original proteinase (chymotrypsin) were then deleted and the new complex saved. SLPI alone was obtained by deleting chymotrypsin from the original structure file. Electrostatic surfaces were generated using SWISSPDB with values of -3.00, 2.00 and 8.00 for the red, white and blue extremes of the spectrum.ResultsThe ELISA confirmed SLPI concentration was fully recoverable at the range of concentrations of NE and CG used in these experiments. However at very high molar ratios of proteinase to inhibitor (> 100:1), there was a fall in quantification which was attributed to the effects of unopposed proteinase activity on the antibody used in the ELISA (Figure 1).Figure 1Validation of the ELISA for measuring SLPI with proteinases. Recombinant human SLPI was mixed with varying concentrations of NE and assayed by ELISA to assess the recovery of SLPI in complex with the proteinase and in the presence of excess proteinase. The molar ratio of proteinase to SLPI is given along the x axis, and the percentage of SLPI recovered is shown on the y axis. The figure shows the results obtained with NE, CG, PPE and trypsin. The ELISA was affected by a large molar excess of proteinase, but not at the molar ratios relevant to this paper (< 10:1).1. Dose-dependent effect of NE on SLPI in A549 supernatantsNE at the concentrations used in these experiments did not affect cell viability (by trypan blue exclusion, data not shown). The concentration of SLPI after 24 h showed a dose related fall with NE treatment from 1.36 ± 0.37 nM at 0.5 nM to 0.20 ± 0.14 nM at 5 nM, whilst media control contained 1.63 ± 0.31 nM (p = 0.006, one way ANOVA) (Figure 2). Similar results were obtained when the experiments were repeated with A549 cells using CG and with PBEC using NE (data not shown).Figure 2Effect of neutrophil elastase (NE) on SLPI in supernatants from cultured A549 cells. A549 cells were cultured for 24 h with NE at the concentrations shown, with an equivalent concentration of the buffer alone, or in a serum free media control. The y axis shows the average of the SLPI secreted in each condition as a percentage of the SLPI secreted by control cells (% SLPI release). Error bars represent standard error of the mean (SEM). N = 3–5 separate experiments for all except buffer control (N = 2). A dose-related reduction in SLPI supernatant concentration was seen with NE (statistically significant from 2.5 nM) (p = 0.006, one way ANOVA).Using a dose range from 0–2.5 nM NE, a linear relationship was seen between the difference in SLPI concentration between treated and control cells, and the amount of NE (Figure 3). This relationship suggests about 1.4 molecules of NE are required to remove 1 molecule of SLPI from cell culture media.Figure 3Relationship between NE and SLPI decrease in the supernatant of A549 cells. The total NE-induced decrease in SLPI concentration, calculated from the experiment shown in Figure 2, was plotted against the concentration of NE used and a linear relationship found (Pearson correlation coefficient 0.846, p < 0.001).2. Effect of other proteinasesAt high concentrations of proteinases, all cells detached from the wells although cell viability was not affected during the duration of the experiment as assessed by trypan blue exclusion (data not shown). At these higher proteinase concentrations, SLPI concentration fell sharply. CG caused a dose-dependent fall in SLPI concentration (79.9 ± 8.2% of control values (p < 0.05) at 1 nM, 4.7 ± 4.3%, 0.02 ± 0.0% and 0.07 ± 0.1%, (p < 0.001) at 10 nM, 100 nM and 1000 nM respectively), but had no effect at 0.1 nM (96.2 ± 0.8% control) while SLPI concentration fell significantly only at high concentrations for PPE (9.0 ± 3.5% at 100 nM, 2.6 ± 1.3% at 1000 nM, p < 0.005) and trypsin (4.5 ± 0.8% at 1000 nM, p < 0.005), at which cell morphology was affected. Mast cell chymase caused a significant reduction in SLPI secretion at concentrations from 10–100 nM. This was associated with a marked change in cell morphology: loss of cell-cell contact but no detachment. At lower concentrations, with normal cell morphology, there was no effect on SLPI secretion. Mast cell tryptase did not affect either SLPI concentration or cell morphology at any dose (Figure 4).Figure 4Effect of other proteinases on SLPI concentration in A549 cells. A549 cells were cultured with varying concentrations of each proteinase for 24 h. The concentration of SLPI, expressed as a percentage of the concentration in serum free media, is shown on the y axis with the SEM represented by error bars. P indicates that the cells exhibited morphological changes of partial detachment, 'rounding up' and losing attachment to each other but remaining adherent to the culture plate, whilst D indicates that the cells detached completely from the culture plate. The effect on SLPI concentration was only independent of cell morphology for CG. Values given are mean ± SEM for at least 3 experiments.3. Time course of the effect of NESeveral groups have demonstrated that the presence of NE reduces the amount of SLPI in cell supernatants after periods of culture ranging from 1 h to 48 h[26,27]. When NE was added to wells containing A549 cells following 24 hours incubation (therefore containing substantial amounts of SLPI), a rapid fall in SLPI concentration was seen at the earliest time point tested (10 min). The results were similar with PBEC (Figure 5a), A549 cells (data not shown) and HepG2 cells (Fig 5b). CG also caused a decrease in supernatant SLPI from media of A549 cells within 10 min (control wells 102.36 ± 3.75% of baseline, wells treated with CG 10 nM 1.50 ± 0.27% baseline at 10 minutes). Subsequent experiments using A549 cells showed that NE already caused a detectable decrease in SLPI within 2 min (data not shown).Figure 5Time course of the effect of NE on SLPI concentration. Figure 5a: PBEC were cultured in basal media for 24 h, and then an aliquot of media was removed as baseline and replaced with the same volume of media control or media containing NE at a concentration sufficient to achieve a concentration of 10 nM in the wells. Further aliquots were taken at the times shown, and SLPI concentration was measured by ELISA. The figure shows the results from 3 experiments. The y axis gives the concentration of SLPI as % of the baseline value for triplicate wells treated with basal media or NE. (Error bars indicate SEM.) The concentration of SLPI fell at the earliest time point studied in wells treated with NE but remained stable in the media controls. Over 24 h the concentration in media controls rose in accordance with the steady state concentrations predicted, but remained low in NE-treated wells. At 24 h, some SLPI was present in the media of NE-treated cells but this was substantially lower than the media controls (p < 0.001 Wilcoxon signed ranks test). b: HepG2 cells were cultured in 12 well plates and, after rinsing with PBS, media containing 1 nM SLPI was added to the cells. After a few minutes equilibration, a baseline aliquot was removed and replaced with either media control or NE to achieve a final concentration of 10 nM. Further aliquots were taken at the time points shown. In the media controls it remained stable throughout, but the concentration in NE-treated wells fell significantly at 10 minutes and remained low thereafter (p < 0.001 Wilcoxon signed ranks test).4. Effect of synthetic NE inhibitorZD0892 alone did not affect SLPI concentration in A549 supernatants (99.12 ± 4.7% of media control). The concentration of SLPI in NE-treated wells fell to 12.05 ± 2% of control values (p < 0.001 compared to media control). When NE and ZD0892 were pre-incubated, the effect of NE on SLPI was abrogated (100.2 ± 3.7% media control, p < 0.005 compared to NE alone). Addition of NE after ZD0892 caused some decrease in SLPI concentration (to 63.0 ± 8.5% of media control, p < 0.05 compared to media control) but the fall in SLPI concentration was less than with NE alone (p < 0.05). Addition of NE before ZD0892 caused a fall in SLPI concentration (to 28.7 ± 13.7% control, p < 0.05 compared to media control), which was not significantly different from NE alone. The difference between the results for NE and ZD0892 added in either order was not statistically significant. These experiments are summarised in Figure 6.Figure 6Timing of the addition of inhibitor to NE affects concentration of SLPI. The diagram shows the concentration of SLPI in the media of wells treated according to the conditions shown on the x axis. The concentration of SLPI is expressed as % of the concentration in control wells. * indicates significant difference from control wells, $ indicates significant difference from NE-treated wells. Values shown are mean ± SEM for 3 experiments. Prior incubation of NE with a tenfold excess of ZD0892 completely abrogated the effect of NE on SLPI concentration. When NE and ZD0892 were added separately, there was a fall in SLPI concentration which was less pronounced than that seen with NE alone, but still significantly different from both media control and NE alone. Although the average fall was greater when NE was added before rather than after ZD0892, this difference was not statistically significant.5. Attachment of SLPI to plastic surfacesWe next explored whether the loss of SLPI in the presence of NE required the presence of cells, or whether it would occur in cell-free conditions. We added SLPI to empty tissue culture plate wells with or without NE. There was a marked loss of SLPI from the empty tissue culture plates as only about 1% of the expected amount was recovered from control wells and therefore an additional effect of NE could not be identified. Further investigation demonstrated that there was a large loss of SLPI when diluted in SFM in standard 20 ml polystyrene tubes, and a further loss in tissue culture plates, which was attributed to charge-related non-specific binding to plastic (data not shown). This may have explained the observed decrease in SLPI in cells treated with high concentrations of trypsin or PPE that caused detachment, since this would have exposed culture plastic which would be capable of binding the previously secreted SLPI, resulting in a fall in SLPI concentration in the supernatant.Use of polypropylene plates and siliconised glass culture systems did not offer more than partial protection against this loss of SLPI (data not shown) and cells did not grow satisfactorily without a substrate on siliconised glass. Addition of HSA 1% w/v to culture media, or coating of tissue culture plates with HSA or human placental collagen prior to plating out cells, partially reduced the non-specific loss of SLPI from culture media (data not shown). Furthermore the presence of HSA in solution partially inhibited the effect of NE in reducing SLPI concentration in cell-free systems (Figure 7), and similarly in plates coated with HSA prior to plating out cells (data not shown). Addition of 1% Tween to cell-free systems completely prevented loss of SLPI when incubated without NE, but did not prevent almost total loss of SLPI in the presence of NE (data not shown).Figure 7Protection by albumin against non-specific binding of SLPI to plastic. The diagram shows the concentration of SLPI in SFM added to tissue culture plates without cells, before addition of NE or control (baseline) and 10 min afterwards. Parallel wells used SFM containing human serum albumin at 1 mg/ml. Data shown are the mean results from 3 experiments with SEM represented by the error bars. Without albumin, almost all the SLPI was lost regardless of whether NE was added. Albumin provided partial protection against loss of SLPI at baseline and after addition of NE.6. Two compartment modelSupernatant from the upper compartments of Transwells on which A549 cells were cultured contained about fourteen times as much SLPI as the lower compartments. The difference may be due to polarized secretion of SLPI, or might be accounted for by SLPI binding to the plastic of the cell culture plates in the lower compartment, which had not been treated to prevent non-specific binding and contained no cells. Addition of NE to the upper compartment caused reduction in SLPI concentration to 10.6 ± 0.3% of control values, and in the lower compartment it caused a reduction to 8.6 ± 1.9% of control. SLPI concentrations in the control-treated compartment were not affected (Figure 8).Figure 8The effect of NE on SLPI concentration in media of cells grown on Transwells. The diagram shows the concentration of SLPI in each compartment of A549 cells grown in Transwells, relative to the concentration in control wells. Wells were treated with either 10 nM NE or serum free media (SFM) alone for 10 minutes, and the concentration of SLPI was then determined by ELISA. Upper and lower compartment results for the same well are adjacent; the solid bars indicate upper compartments and the open bars indicate lower compartments. SFM/NE indicates that NE was applied to the lower compartment only whilst NE/SFM indicates that NE was applied to the upper compartment only. The concentration of SLPI in the NE-treated compartments fell significantly (p = 0.001 by independent t-test) from 100% to 10.6 ± 0.3% (NE added to upper compartment) and 8.6 ± 1.9% (NE added to lower compartment) whilst there was no difference in concentration of the paired SFM-treated compartments (99.9 ± 1.5% and 90.31 ± 7.8% of control wells respectively).7. Theoretical modelling of SLPI in complex with proteinasesFigure 9 shows the SLPI molecule alone (Fig 9a) and SLPI-proteinase complexes (Fig 9b–f). Using colour to indicate charge (red for negative, blue for positive), it can be seen that both NE and CG produced a highly positively-charged complex with SLPI. Mast cell chymase and trypsin varied in charge on the two aspects shown whereas mast cell tryptase was highly negatively-charged on both aspects. PPE was not modelled for this experiment because it does not bind SLPI. The results suggest that SLPI in complex with CG and NE would be predicted to associate more with negatively-charged surfaces than either free SLPI, or SLPI complexed with other proteinases, because of the greater positive charge.Figure 9Predicted charge of the complex of SLPI with various proteinases. Modelling of the SLPI molecule alone (A) and in complex with various proteinases (B-F): blue indicates positive charge, white is neutral and red indicates negative charge. All models are shown in the same orientation with views of opposite sides. The SLPI molecule was predominantly neutral with some positively-charged areas. Cathepsin G (D) formed the most positively-charged complex, followed by NE (C). Chymase (F) was strongly positively-charged on one surface only. Trypsin (B) had weak positive and negative charge on different surfaces, and tryptase (E) was strongly negatively-charged.DiscussionThe results from the present study show that both NE and CG decrease SLPI released into culture supernatants from lung epithelial cells. The dose response of the proteinases was linear, and the effect was almost immediate. Experiments using a synthetic NE inhibitor showed that when the inhibitor was added together with NE, the NE-mediated decrease in SLPI was completely prevented. In contrast, when the inhibitor was added after NE had been applied to the cells it only partially prevented a reduction in SLPI. Experiments using a Transwell model showed that the effect of NE on SLPI required direct contact between these molecules, suggesting that direct binding of NE or CG to SLPI was involved.Only the strongly cationic proteinases NE and CG were able to reduce SLPI concentration at almost molar equivalent doses. The other proteinases only caused reduction in SLPI when used at concentrations sufficient to alter cell morphology and/or cause detachment, which probably leads to non-specific binding to the tissue culture plastic. Theoretical modelling of surface charge supported the concept that an NE-SLPI or CG-SLPI complex would have a greater positive charge than SLPI alone or in complex with the other proteinases studied. Therefore it seems likely that the reduction in SLPI concentration in the presence of proteinases was mediated by charge-related binding to other structures.Previous studies on the effect of NE on SLPI in vitro have shown a decrease in SLPI concentration with a variety of cells and tissues [25-27], but only two studies have attempted to explore this further. The study by Marchand et al[28] used immunohistochemistry to examine nasal explanted epithelial tissue exposed to NE and found greater signal than in the control tissues not exposed to NE. The study by van Wetering et al[27] used PBEC and again found a dose-dependent effect, present from a few hours after exposure of cells to NE, which could be inhibited by co-administration of an inhibitor (α1 AT). Analysis of cell lysates indicated that SLPI became cell-associated in the presence of NE rather than in the culture media, and hence suggested a block in secretion or redistribution of the protein rather than an effect on synthesis. These studies did not pursue more detailed analysis of the relationship between NE and SLPI concentration and the possible mechanism responsible for this relationship. In addition, none of the studies in the literature has controlled for non-specific binding of SLPI to tissue culture materials. Studies on the ability of SLPI to protect fibronectin from degradation by NE showed that SLPI could associate with this substrate through ionic interactions [48] and examination of human lung tissue showed that SLPI was associated with elastin fibres[49], again suggesting non-specific binding. SLPI binds non-specifically to many large molecules such as mucins[50] and DNA[51], however, to our knowledge no studies have examined whether the NE-SLPI complex is more likely to associate with cells or substrates than SLPI alone.Our initial validation studies demonstrated that in the presence of a molar excess of NE or CG, detection of SLPI by ELISA was not affected. The studies described here did not examine the possibility of cleavage or degradation of SLPI by excess of proteinase, but even if this happened, the cleavage products would appear to be detected fully by the ELISA, suggesting that the monoclonal capture antibody recognises an epitope not affected by proteolysis, and that the polyclonal detection antibody also recognises preserved sites. Incubation with samples containing excessive amounts of NE may however expose the capture antibody to proteolytic degradation, and this may be the explanation for the fall in detection at high concentrations of NE, rather than degradation of SLPI by NE. Since the validation studies used recombinant rather than endogenous SLPI and were conducted in assay buffer rather than conditioned cell culture media, it cannot be guaranteed that the ELISA would perform as reliably in experimental conditions. However in the published studies previously mentioned, the same effect of dose-dependent reduction of SLPI in the cell culture environment was shown by systems using a variety of different antibodies for detection of SLPI, suggesting that the effect is real rather than a systematic measurement error. Possible effects on the ELISA from complex formation or cleavage of SLPI therefore cannot fully explain the complete loss of signal of SLPI in the presence of cells treated with NE. Nevertheless, our experiments do not conclusively demonstrate that SLPI bound to cells in presence of NE is biologically active. Furthermore, it needs to be noted that the difficulties in avoiding non-specific binding of SLPI to the plastic components of the cell culture system are not fully countered in these studies. If the hypothesis that binding of SLPI to NE causes adherence to cells by charge interactions is valid, then the same mechanism could apply to any negatively-charged surface, such as plastic cell culture components which were also shown to bind free SLPI avidly. NE did appear to increase non-specific binding of SLPI to plastic, because when detergent was used as a blocking agent, SLPI concentration fell only in the presence of NE (suggesting that the higher ionic charge of the NE-SLPI complex was able to overcome the concentration of detergent used). The ELISA system contains strong buffering agents that must be presumed to protect against loss of free SLPI. The validation studies suggest that they are sufficient to protect also against loss of the NE-SLPI complex. However it cannot be presumed that there was not loss of SLPI to plastic during the cell culture process, harvesting of supernatant and storing of the samples prior to preparation of the ELISA, and that this loss of SLPI was not enhanced in the presence of NE solely by increased binding to plastic rather than to cell membranes. Proof of the concept of binding of SLPI to cells would require a different experimental approach, and therefore the experiments presented here cannot claim to prove that the effect is biological and relevant to the in vivo situation. The location of the lost SLPI protein remains uncertain, although preliminary immunofluorescence studies in our laboratory suggested at least in part that the SLPI does become internalised (data not shown). Further studies, using SLPI bound to a fluorescent or radioactive label that does not impede binding to NE, will be required to confirm this.There is a likely biological relevance of the phenomenon of free SLPI associating with cell membranes and extracellular matrix proteins because this would allow the lung to establish a protective antiproteinase coat over structures that would be at most risk of damage by free NE. The major function of NE is probably intracellular killing of bacteria within the phagolysosome. Where neutrophils are migrating through the lung, some local NE activity (from a released azurophil granule or released from cell membrane association) may possibly be beneficial to this process. Free NE in lung secretions however is believed to be uniformly deleterious. Where this occurs, it would be neutralized to some extent by the presence of SLPI bound to vulnerable structures. There may be some value in the SLPI-NE complex associating with these structures. If the NE-SLPI complex prevented further association of NE with extracellular matrix substrates more effectively than SLPI alone (because of its higher positive charge), it would leave the free NE in epithelial lining fluid to be inactivated by α1 AT, which the latter cannot do once NE is bound to substrate[52]. There are no published data on the avidity of binding of the NE-SLPI complex to cell membranes or matrix substrates. However it is known that there is a strong association between NE and neutrophil cell membranes[30], and between SLPI and fibronectin[48], based on the ionic strength or pH required to dissociate them. The ionic charge of the NE-SLPI complex has never been measured, because the binding of these two strongly cationic molecules is sufficiently reversible that standard electrophoretic methods cause them to dissociate. It is also unknown how stable the complex would be when membrane-bound. It is possible that membrane-bound complex might be able to dissociate, or be passively internalised during membrane cycling, or actively internalised by endocytosis or phagocytosis, probably requiring a specific receptor. Internalisation of the complex might generate biological responses central to lung inflammation. Further studies will clearly be required to explore these possibilities.ConclusionIn summary, the experiments described in this paper support the hypothesis that the association of SLPI in complex with NE to negatively-charged structures leads to its removal from cell culture media, and that this effect is a passive biochemical process, probably dependent on charge. It is likely that this effect occurs with cell membranes as well as non-biological components, but this has yet to be confirmed. Future experiments should include measures to counter the ability of SLPI to bind non-specifically to a variety of structures, particularly in the presence of NE or CG.AbbreviationsSLPI: Secretory leukoproteinase inhibitor; NE: Neutrophil elastase; COPD: Chronic obstructive pulmonary disease; CB: Chronic bronchitis, α1 AT: alpha 1 antitrypsin inhibitor; CG: Cathepsin G, PPE: Porcine pancreatic elastase; ELISA: enzyme linked immunosorbent assay; ECACC: European Collection of Cell Cultures; DMEM: Dulbecco's modified Eagle's medium; FCS: Fetal calf serum, ATCC: American Type Culture Collection; MEM: Minimum essential medium; PBEC: primary bronchial epithelial cells; PBS: Phosphate-buffered saline; SEM: Standard error of the mean; SFM: Serum free media, HSA: Human serum albumin, DNA: deoxyribonucleic acid.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsAS carried out the experimental work and wrote the manuscript. TD carried out the molecular modelling studies and produced the illustrations. PH conceived the hypothesis, advised on experimental work and assisted in drafting the manuscript. RS supervised the experimental work and assisted in drafting the manuscript. All authors read and approved the final manuscript.Availability & requirements\n\nREFERENCES:\nNo References"
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+{
+    "id": "PMC2529324",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2529324\nAUTHORS: Shih-Yen Ku, Yuh-Jyh Hu\n\nABSTRACT:\nBackgroundStructural similarities among proteins can provide valuable insight into their functional mechanisms and relationships. As the number of available three-dimensional (3D) protein structures increases, a greater variety of studies can be conducted with increasing efficiency, among which is the design of protein structural alphabets. Structural alphabets allow us to characterize local structures of proteins and describe the global folding structure of a protein using a one-dimensional (1D) sequence. Thus, 1D sequences can be used to identify structural similarities among proteins using standard sequence alignment tools such as BLAST or FASTA.ResultsWe used self-organizing maps in combination with a minimum spanning tree algorithm to determine the optimum size of a structural alphabet and applied the k-means algorithm to group protein fragnts into clusters. The centroids of these clusters defined the structural alphabet. We also developed a flexible matrix training system to build a substitution matrix (TRISUM-169) for our alphabet. Based on FASTA and using TRISUM-169 as the substitution matrix, we developed the SA-FAST alignment tool. We compared the performance of SA-FAST with that of various search tools in database-scale search tasks and found that SA-FAST was highly competitive in all tests conducted. Further, we evaluated the performance of our structural alphabet in recognizing specific structural domains of EGF and EGF-like proteins. Our method successfully recovered more EGF sub-domains using our structural alphabet than when using other structural alphabets. SA-FAST can be found at .ConclusionThe goal of this project was two-fold. First, we wanted to introduce a modular design pipeline to those who have been working with structural alphabets. Secondly, we wanted to open the door to researchers who have done substantial work in biological sequences but have yet to enter the field of protein structure research. Our experiments showed that by transforming the structural representations from 3D to 1D, several 1D-based tools can be applied to structural analysis, including similarity searches and structural motif finding.\n\nBODY:\nBackgroundGenome sequencing projects continue to produce amino acid sequences; however, understanding the biological roles played by these putative proteins requires knowledge of their structure and function [1]. Despite that empirical structure determination methods have provided structural information for some proteins, computational methods are still required for the large number of proteins whose structures are difficult to determine experimentally. And while the primary sequence should contain the folding guide for a given protein, our ability to predict the three-dimensional (3D) structure from the primary sequence alone remains limited. Some ab initio methods do not require such information, but the application of these methods is often limited to small proteins [2,3].Structure alignment research has led to the discovery of homologues of novel protein structures. And, although many structure alignment tools have been developed, such as CE [4], DALI [5], VAST [6], MAMMOTH [7], FATCAT [8], and Vorolign [9], we wanted to provide a different perspective on protein structure analysis. Previous studies of protein structures have shown the importance of repetitive secondary structures, particularly α-helices and β-sheets, in overall structure determination. Together with variable coils, these structures constitute a basic three-letter structural alphabet that has been used in the development of early-generation secondary structure prediction algorithms (such as GOR [10]) as well as more recent-generation algorithms. These newer algorithms have been applied to neural networks, homology sequences, and discriminative models [11-14], and their accuracy in predicting secondary structure approaches 80%. However, despite this predictive accuracy, the three-letter alphabet does not contain the information necessary to approximate more refined 3D reconstructions.The recent rapid increase in the number of available protein structures has allowed more precise and thorough studies of protein structures. Several authors have developed more complex structural alphabets that incorporate information about the heterogeneity of backbone protein structures by using subsets of small protein fragments that are observed frequently in different protein structure databases [15-17]. The alphabet size varies from several letters to about 100 letters [18]. For example, Unger et al. [19] and Schuchhardt et al. [20] used k-means methods and self-organizing maps (SOMs), respectively, to identify the most common folds, but the number of clusters generated was too large to have substantial predictive value. By applying autoassociative neural networks, Fetrow et al. defined six clusters that represent super-secondary structures which subsume the classic secondary structures [21]. Bystroff and Baker produced similar short folds of different lengths and grouped them into 13 clusters that they used to predict 3D structure [22]. Camproux et al. developed a hidden Markov model (HMM) approach that accounted for the Markovian dependence to learn the geometry of the structural alphabet letters and the local rules for the assembly process [23]. Fixing the alphabet size to 23 letters, Yang & Tung applied a nearest-neighbor algorithm on a (κ, α)-map of structural segments to identify the 23 groups of segments used in their alphabet [24]. More details about these local structures can be found in a recent review [25].In this study, we developed a flexible pipeline for protein structural alphabet design based on a combinatorial, multi-strategy approach. Instead of applying cross-validation [22] or Markovian processes [15] to refine the clusters directly, we used SOMs and Bayesian Information Criterion (BIC) to determine the optimum size of structural alphabet. We then applied the k-means algorithm [26] to group protein fragments into clusters, forming the bases of our structural alphabet. Moreover, unlike most other works that built substitution matrices for alphabets based on known blocks of aligned proteins, we used a matrix training framework that generated matrices automatically without depending on known alignments. An expressive structural alphabet allows us to quantify the similarities among proteins encoded in the appropriate letters. It also enables the primary representation of 3D structures using standard 1D amino acid sequence alignment methods. To demonstrate the feasibility of our new method, we verified the application of the alphabet produced by our pipeline and the trained substitution matrix to a widely used 1D alignment tool, FASTA [27]. We conducted several experiments using the same datasets used in other recently published works and evaluated the performance of our tool in database-scale searches. In addition to investigating whether our alphabet and matrix worked well with 1D alignment tools in database searches, we evaluated the ability of our structural alphabet to characterize local structural features.ResultsStructural alphabetBy combining SOMs, minimum spanning trees, and k-means clustering, we developed a multi-strategy approach to designing a protein structural alphabet. To derive an appropriate substitution matrix for the new alphabet, we developed a matrix training framework that would automatically refine an initial matrix repeatedly until it converged. Unlike some previous works that presumed the size of the alphabet [23], our method determined the alphabet size autonomously and statistically. Various experiments were conducted to evaluate our methodology.The SOM is an unsupervised inductive learner and can be viewed as topology preserving mapping from input space onto the 2D grid of map units [28]. The number of map units in SOMs defines an inductive bias [29], as does the number of hidden units for the feedforward artificial neural networks, and it affects the clustering results. By systematically varying the number of SOM map units and applying BIC, we identified the most frequent number of clusters that maximized the BIC and used this number to define the size of the alphabet. We tested SOMs ranging in size from 10 × 10 to 200 × 200, ultimately defining the size of our alphabet at 18 letters. The relationship between number of clusters found and number of SOM map units used is summarized in Table 1.Table 1Relationship between the number of clusters found and the number of SOM map units usedSOM map sizeNumber of clustersSOM map sizeNumber of clusters10 × 106110 × 1102420 × 209120 × 1201930 × 3010130 × 1302140 × 4012140 × 1402250 × 5015150 × 1501860 × 6013160 × 1601570 × 7014170 × 1702180 × 8018180 × 1801890 × 9018190 × 19018100 × 10020200 × 20018Our analysis determined that among the number of clusters that maximized the BIC, 18 clusters occurred most frequently. Thus, we assigned 18 letters to our alphabet.To verify whether fragments were assigned to the same cluster by the various SOMs, we analyzed those SOMs (with varying numbers of map units) that produced 18 clusters, including SOMs sized 80 × 80, 90 × 90, 190 × 190 units, etc. We calculated the overlap level between any two of the SOMs, defined as percentage of fragments that belonged to the same cluster. The average overlap between all pairs of SOMs for each of the 18 clusters was over 90%, indicating that these clusters were very consistent (Table 2). Table 3 and 4 display the within-cluster Euclidean distance, defined as the average distance of each segment to the center, and the center-to-center Euclidean distance for the 18 protein fragment clusters found by our method and by SOM alone, respectively. The average Phi/Psi angles (i.e. the Phi/Psi angles of the centroid) for the 18 clusters are presented in Table 5. As indicated in Table 3 and 4, the within-cluster Euclidean distances for our clusters were smaller than those of the SOM clusters, which suggested that our 18 clusters were more coherent. On the other hand, the center-to-center distances for our clusters were larger than those of the SOM clusters, indicating that our clusters were better separated from each other. The 3D conformation of the representative segment for each alphabet letter is illustrated in Figure 1 and the superimposition of protein segments is shown in Figure 2. To verify that these representative segments could be the building blocks for protein structures, we analyzed the frequency of their occurrence in four major structural classes according to the Structural Classification of Proteins (SCOP): all-alpha, all-beta, alpha/beta, and alpha+beta [30]. The frequency of each category of segments is presented in Table 6. The alpha helix segments represented by alphabet letters T, P, and R occurred more often in the all-alpha class than did the other segments. Similarly, more beta sheet segments, such as N, E, and A, were found in the all-beta class. In both the alpha/beta and alpha+beta classes, most of the segments were found to be either alpha helices or beta sheets.Table 2The average overlap between all pairs of SOMs that produced 18 clusters of fragmentsCluster123456789101112131415161718Overlap99.898.496.797.497.494.399.195.097.894.699.895.696.795.395.798.296.395.5Table 3Summary of the within-cluster Euclidean distance and the center-to-center Euclidean distance for 18 protein fragment clusters found by our alphabet design pipelineWithin-ClusterCenter-to-CenterMeanSD1817161514131211109876543211116.637.2252.3300.4330.1242.8181.7182.1317.6327.7415.4266.3329.0181.7242.5262.2273.6253.4193.202238.738.5315.8226.6272.7197.4243.3227.2285.3270.5346.1283.9285.4261.3189.5182.3215.0296.003264.729.8219.7279.8193.6220.6190.4284.1251.1292.9413.2195.1237.6181.4324.4234.1285.904319.341.5297.8297.0270.7285.5311.5288.6286.9317.1352.2302.9184.3250.7256.2193.305250.439.7248.6268.9190.2238.1302.2280.1258.5287.2406.6267.2258.8192.8229.006257.528.0220.4174.2242.3180.4262.8266.2264.4229.1310.3322.3270.9308.60772.220.4220.8356.7289.2297.5266.1244.8307.2361.1478.3248.9316.808282.231.0275.3214.2186.1218.9259.1335.6258.2253.8286.9273.909320.927.9275.8287.6250.7244.5222.6292.2286.7307.3354.3010148.826.1406.3243.1334.4286.3333.5361.8293.2240.801197.143.4290.4169.5214.8178.9248.7238.3270.4012272.032.7259.7226.6200.7218.7269.1325.6013133.633.2291.2309.3334.3267.6230.5014272.831.4255.5206.2258.7145.3015106.232.3241.176.8162.1016109.039.1221.8172.601733.223.2272.9018146.238.20Table 4Summary of the within-cluster Euclidean distance and the center-to-center Euclidean distance for 18 protein fragment clusters found by the SOM aloneWithin-ClusterCenter-to-CenterMeanSD1817161514131211109876543211129.138.9220.9270.9302.4202.7175.7161.9277.2295.9381.4233.8307.6181.6234.8223.0263.6250.5164.402242.739.4304.8198.1265.0192.0201.2217.5241.3237.1309.9244.0247.9259.6165.5169.5189.0273.503265.829.8180.3276.0168.3177.9169.5266.4237.3256.2397.2185.0218.6156.4298.5224.5280.404327.141.4261.7275.8265.8241.5298.8273.8250.9297.5321.2266.9182.9215.2250.4156.805251.939.6206.8223.7150.2207.1300.9258.4217.8274.8400.7227.2243.6167.1227.606260.729.2202.0158.5235.8137.3248.4225.3258.8205.8304.7300.23235.0297.00775.720.5191.9323.9243.6280.5238.4199.0292.7346.6463.2247.1291.308291.430.8250.4196.2144.8203.1245.8322.9245.4226.8265.6272.709329.127.9275.3251.6219.3200.6197.1263.8278.1272.5342.3010157.927.4364.8240.4310.3262.0292.9329.8266.4234.3011113.845.8244.9156.7190.2167.5224.8213.6254.9012283.032.4215.7205.4197.6191.5239.0299.2013170.329.5277.6272.6322.4252.2188.8014277.832.6238.5179.7239.899.5015111.233.1210.659.5161.6016114.0538.4219.4146.801736.224.8228.6018158.537.40Table 5The average Phi/Psi angles (i.e. the Phi/Psi angles of the centroid) for the 18 clusters found by our alphabet design pipelineΦ(i)Φ(i+1)Φ(i-1)Φ(i+2)ψ(i)ψ(i-1)ψ(i-2)ψ(i+1)1(A)-97.99-70.43-104.52-79.77132.99118.98132.37-44.262(R)-67.81-67.48-92.52-69.17-52.78134.7596.12-35.693(N)-98.66-99.17-83.46-104.16132.5675.64-36.97134.014(D)90.39-63.35-93.54-84.31-5.4397.71115.2294.645(C)-88.09-102.50-93.58-97.49-51.5688.66106.12133.276(Q)-65.87-69.19-85.50-59.89-35.12-50.41129.98-37.577(E)-107.28-96.08-107.66-105.96132.71130.92133.88133.068(G)89.16-93.43-62.92-90.2520.650.22-32.5085.949(H)-91.05-90.1691.91-91.53100.48103.365.4075.5610(I)58.5956.7955.5054.75-42.38-38.76-47.77-48.4611(L)-71.08-84.21-65.9287.57-21.11-29.95-31.8020.0012(K)-83.0795.78-69.02-91.349.50-9.18-5.50100.5213(M)-88.72-64.82-95.7291.27100.65113.69107.430.7014(F)-87.36-71.63-75.80-68.31134.6958.97-35.87-49.7215(P)-96.95-78.84-75.71-78.034.072.17-33.25-25.9216(S)-83.07-95.71-63.62-97.87-28.27-28.59-38.35126.5717(T)-63.55-65.43-62.97-68.03-42.53-41.88-42.16-38.3418(W)-105.06-91.96-78.47-94.14122.89-83.40109.6499.64Table 6Frequency of occurrence of the protein segments defined by our alphabet in four main SCOP classesAll alphaAll betaalpha/betaalpha+betaLetterCountPercentageCountPercentageCountPercentageCountPercentageA548592.952554738.832782385.461610416.07R913634.921483615.132703455.311456195.49N761764.1030983410.713406826.692025557.64D210551.131271594.391120782.20669592.53C348561.881723345.961939523.811026323.87Q1024445.511113333.852718935.341380815.21E586723.1678260727.0662071712.1842777816.14G423502.28721052.491473902.89769682.90H390172.101155423.991633193.21892033.37I35470.1966070.2394490.1957390.22L493122.65409091.411416052.78658562.48K435822.35586872.041468692.88705492.66M167270.901270704.391103182.17679122.56F707183.81893663.091791453.52917023.46P1043645.62549391.911926543.78871493.29S760804.10837252.891739353.41911603.44T93793850.491492595.17155158530.4665152524.58W345331.861864766.461900013.721084604.09Total1857593100.002891786100.005094175100.002650888100.00Figure 1The 3D conformation of the representative segment for each alphabet letter.Figure 2Superimposition of protein segments in the 18 clusters.TRISUM – Substitution matrixMost approaches to constructing substitution matrices require the alignment of known proteins [24,31,32]. Because alignments are not always available and their validity can be dubious, we used a self-training strategy to build the substitution matrix for our new structural alphabet. This training framework had a flexible and modular design, and unlike most other approaches, it did not rely on the pre-alignment of protein sequences or structures. Different training data or alignment tools can be incorporated into this framework to generate appropriate matrices under various circumstances. In this study, we used the non-redundant proteins contained in SCOP1.69 with sequence similarity of less than 40% for training, excluding those proteins in SCOP-894 and the 50 test proteins (see details below) to ensure that the training data and the testing data did not overlap. We defined the positive hit rate of a query as the ratio of the number of positive hits to the size of the family the query belonged to. As we iterated each training protein (as a query), we refined the matrix until we could no longer increase the average positive hit rate of all the proteins. We tried different learning rates ranging from 0.25 to 1.00. The final average positive hit rates under different learning rates were similar, ranging between 0.9112 and 0.9153. An example of the learning curve of matrix training is presented in Figure 3. We selected the converged matrix with the maximum positive hit rate with the learning rate set to 0.50. We named this matrix TRISUM-169 (TRained Iteratively for SUbstitution Matrix-SCOP1.69), as shown in Figure 4.Figure 3Example learning curve of matrix training. The average positive hit rate converged at 0.9153 with the learning rate set to 0.5.Figure 4The substitution matrix TRISUM-169.Comparison with other toolsSeveral protein structure search tools based on 1D alignment algorithms have been developed, including SA-Search [33], YAKUSA [34], and 3D-BLAST [24]. Yang and Tung tested 3D-BLAST on the SCOP database scan task [24]. They prepared a protein query dataset named SCOP-894 from SCOP 1.67 and 1.69; this dataset contains 894 proteins with <95% sequence similarity. We tested SA-FAST on the same dataset in order to allow direct comparison (Table 7). The results indicated that SA-FAST outperformed 3D-BLAST and PSI-BLAST in the test of the SCOP-894 query dataset.Table 7SA-FAST versus 3D-BLAST and PSI-BLAST in SCOP structural function assignment accuracy for the SCOP-894 protein datasetClass894 proteins Accuracya (894 proteins)Accuracy (sequence identity <25%)Number of queriesSA-FAST3D-BLASTPSI-BLASTSA-FAST3D-BLASTPSI-BLASTAll alpha16199.2794.4194.4195.8375.0066.67All beta19995.1294.4793.9787.3277.5573.33α/β29297.5897.2691.4495.6887.8865.77α+β24295.1394.6388.8493.8183.3360.87aThe top-ranked family in the hit list of a query was used as the predicted family. Accuracy is the percentage of times that the family was correctly predicted.We also used the same 50 proteins selected from SCOP95-1.69 that were used by Yang & Tung to compare SA-FAST with 3D-BLAST, PSI-BLAST, YAKUSA, MAMMOTH, and CE, in search time, predictive accuracy, and precision. Other search tools exist, such as PBE [35], SA-Search [33], and Vorolign [9], but because they either could not be tested on the SCOP database directly or the versions of their databases provided were too old (e.g. ASTRAL in PBE derived from SCOP-1.65, Vorolign server only scans SCOP40-1.69), these tools were not used in the comparisons. The results showed that SA-FAST outperformed the other two BLAST-based search tools (i.e. 3D-BLAST and PSI-BLAST) and another structure search tool that describes structures as 1D sequences (YAKUSA) in both predictive accuracy and precision (Table 8). Additionally, SA-FAST was comparably accurate and precise as the structural alignment tools MAMMOTH and CE. Regarding search time (using one Intel Pentium 2.8 GHz processor and 512 Mbytes of memory), Table 8 clearly indicates that SA-FAST was far more efficient than were the structural alignment tools MAMMOTH and CE.Table 8Comparison between SA-FAST, 3D-BLAST, PSI-BLAST, YAKUSA, MAMMOTH, and CE on 50 proteins selected from SCOP95-1.69Search toolAverage time required for a query (sec)Relative to SA-FASTAccuracya (%)Average precisionb (%)SA-FAST1.151.009690.803D-BLAST1.301.139485.20PSI-BLAST0.480.428468.16YAKUSA8.887.729074.86MAMMOTH1834.181594.9410094.01CE22053.3219176.809890.78aThe top-ranked family in the hit list of a query was used as the predicted family. Accuracy is the percentage of times that the family was correctly predicted.bThe precision is defined as T/H, where T is the number of true hit structures in the hit list, and H is the total number of structures in the hit list.To further evaluate the predictive validity of our alphabet, we examined pairwise alignment of difficult cases based on the number of residues aligned and the superposition root mean square deviation (RMSD). To avoid alignment process bias and to maintain consistency in our analysis of various structural alphabets, we applied the same FASTA-based alignment algorithm [27] in the alignment tests. We tested the alphabets and substitution matrices used in PBE-align, 3D-BLAST, and SA-FAST on ten difficult cases of previously studied pairwise alignments and compared the results with those produced using VAST, DALI, CE, and FATCAT [8,36]. Based on the alignments obtained using different alphabets and matrices, we used VMD [37] to calculate the superposition RMSD for PBE-align, 3D-BLAST, and SA-FAST. Table 9 shows that our alphabet had the lowest average RMSD per aligned residue among the three structural alphabets in the ten difficult alignment tests. Figure 5 shows four superimposition examples based on our structural alphabet.Table 9Results of ten difficult cases of pairwise alignmentProtein 1Protein 2VASTDALICEFATCATYang & Tung'sde Brevern et al.'sOur SA1fxia1ubq48(2.10)60(2.60)64(3.80)63(3.01)59(2.76)76(2.89)58(2.64)1ten3hhrb78(1.60)86(1.90)87(1.90)87(1.90)57(2.57)73(2.31)90(2.24)3hlab2rhe_-63(2.50)85(3.50)79(2.81)54(2.65)78(3.01)79(2.87)2azaa1paz_74(2.20)81(2.50)85(2.90)87(3.01)70(2.34)57(2.23)87(2.40)1cewi1mola71(1.9)81(2.30)69(1.90)83(2.44)52(2.37)53(2.35)61(1.83)1cid_2rhe_85(2.20)95(3.30)94(2.70)100(3.11)54(2.75)53(2.49)55(2.08)1crl_1ede-211(3.40)187(3.20)269(3.55)167(3.35)120(3.47)187(3.25)2sim_1nsba284(3.80)286(3.80)264(3.00)286(3.07)121(2.75)121(2.96)137(3.2)1bgea2gmfa74(2.50)98(3.50)94(4.10)100(3.19)27(3.34)77(2.8)78(2.72)1tie_4fgf_82(1.70)108(2.00)116(2.90)117(3.05)91(3.15)62(3.45)115(3.05)Average RMSD/aligned-residues0.02260.02380.02610.02290.03730.03630.0278The number of residues aligned and the RMSD (in parentheses) are shown. The last row displays the average RMSD per aligned residue. Except for PBE-align, 3D-BLAST, and SA-FAST, the results of the methods were adopted from [36].Figure 5Superimposition examples based on alignments identified by SA-FAST. (a) 1fxiA & 1ubq_ (b) 2azaA & 1paz_ (c) 1cewI & 1molA (d) 1cid_ & 2rhe.Local structure conservation in putative active sites can reflect biological meaning and these types of structural patterns can be used to predict protein function [19], e.g., the binding sites for metal-binding proteins [38]. Conserved local structural features can be identified in various ways and described using different representations. Because of the aforementioned advantages to 1D representation, we wanted to evaluate the feasibility of describing structural domains/sub-domains using our structural alphabet. Because there is no motif finding tool specifically designed for protein structural alphabets, we applied the motif finding programs available to evaluate the feasibility of using structural alphabets to characterize local structure features. Currently, we use the motif finding program, MEME [39] to identify common structural motifs in protein families. We tested our method on a well-known protein family, the epidermal growth factor (EGF)/EGF-like family. Based on the information published in literature or recorded in databases, we could verify whether the protein domains/sub-domains in EGF/EGF-like proteins could be described accurately using structural alphabets. EGF domains comprise extracellular protein modules described by 30–40 amino acids primarily stabilized by three disulfide bonds. Homologies and functional data suggest that these domains share some common functional features. If we number the cysteine residues as Cys1 to Cys6, where Cys1 is the closest to the N-terminus, the regularity of cysteine spacing defines three regions: A, B, and C. Based on the conservation in sequence and length of these regions, the homologies have been classified into three different categories [40]. We first described the 227 proteins in the EGF-type module family of SCOP 1.69 using our alphabet and the alphabets of Yang & Tung's [24] and de Brevern et al. [16,35]. We then used MEME to identify the common motifs corresponding to the A, B, and C sub-domains. According to InterPro [41], 24 of these proteins were exclusively of EGF Type-1, 74 were of EGF-like Type-2, and 117 belonged to EGF-like Type-3 only. We classified the remaining 12 proteins as Others. Sub-domain A was typically composed of five to six residues in Types 1 and 2, sub-domain B usually contained 10–11 residues in Type-1 but was consistently three residues shorter than in Type-2. Sub-domain C was conserved in length and contained four or five specific residues in Type-1 and Type-2 [40]. The sub-domains in EGF-like Type-3 were less conserved. A found motif was considered to correspond to a sub-domain if more than one-half of the residues in the sub-domain were included in the motif. If any single motif correctly corresponded to a sub-domain, we claimed that this sub-domain was recovered successfully (that is, a hit). The results of the motifs found are summarized in Table 10 and 11. They show that MEME was able to identify more EGF sub-domains using our structural alphabet than using the alphabets of Yang & Tung or de Brevern et al. One example of each EGF group is shown in Figure 6, including the structures with highlighted sub-domains. Using our alphabet, MEME identified meaningful motifs that covered all three sub-domains in the EGF examples (Figure 6); however, using Yang & Tung's or de Brevern et al.'s alphabets, the motifs found covered only one or two sub-domains.Table 10Comparison between our structural alphabet (used in SA-FAST) and those of Yang & Tung (used in 3D-BLAST) and de Brevern et al. (converted by PBE-T, a facility associated with PBE-align) for describing motifs found by MEME within the EGF familyOur SAYang & Tung'sde Brevern et al.'sSub-domain TypeABCABCABCEGF proteinsNo.aHitsbCovcHitsCovHitsCovHitsCovHitsCovHitsCovHitsCovHitsCovHitsCovType 1242395.82291.72395.81145.82187.51979.21875.01458.31875.0Type 2747398.67195.974100.06283.87398.66081.16891.96283.87094.6Type 311711699.110690.66152.15446.210287.22521.410993.211295.74841.0Others1212100.01191.71191.7975.01191.7975.012100.01191.7975.0All22722498.621092.516974.413659.920791.211349.820791.219987.714563.9aThe number of EGF proteins of a specific type, bA hit for a sub-domain occurred when more than half of the sub-domain residues were contained in a given motif. We present the number of hits of different types, cCov(Coverage) was defined as the ratio of the number of hits to the number of EGF proteins, e.g., if No. = 24 and Hits = 22, then Cov = 22/24 = 91.7%.Table 11Statistical analysis of EGF(EGF-like) proteins whose sub-domains were detected by MEMEStructural AlphabetEGF proteinsOur SAYang & Tung'sde Brevern et al.'sCountPercentageCountPercentageCountPercentageFound 3a15166.527934.8010445.81Found 2b7432.607834.3611651.10Found 1c20.886327.7573.08Found 0d00.0073.0800.00Total227100.00227100.00227100.00aEGF (EGF-like) proteins in which all three sub-domains (A, B and C) were found by MEME, bEGF (EGF-like) proteins in which two of the three sub-domains were found by MEME, cEGF (EGF-like) proteins in which only one sub-domain was found by MEME, dEGF (EGF-like) proteins in which MEME failed to identify any sub-domain.Figure 6Examples of structural motifs corresponding to EGF sub-domains. We colored the sub-domains A, B, and C in blue, green, and red, respectively. The motifs that corresponded to EGF sub-domains, using our structural alphabet and those of Yang & Tung and de Brevern et al., were also highlighted in blue, green, and red. The overlapping region between motifs was colored purple. In the sequence view, the first three sequences are EGF protein represented by our structural alphabet, the alphabet of Yang & Tung, and the alphabet of de Brevern et al., respectively. The fourth is the amino acid sequence with the cysteines highlighted in orange. The sub-domains are marked at the bottom.DiscussionThis study aimed to: (1) introduce a systematic and modular pipeline for protein structural alphabet design, and (2) analyze the potential of our new alphabet to characterize local protein properties. There are two features that distinguish our method from the others. First, we took a multi-strategy approach to structural alphabet design. The alphabet size was automatically and statistically determined based on BIC and was visualized using a unified distance matrix (U-matrix). We did not pre-specify the alphabet size [24] or use an ad hoc procedure, such as iterative shrinking, to find the optimal size [15]. And, unlike other methods that use specialized databases, e.g. Pair Database [24] and PDB-SELECT [32,42], the protein structure data used to build the alphabet were obtained from the non-redundant PDB (nrPDB) database and were not pre-processed for any particular purpose, ensuring the generality of our alphabet. Second, we proposed a novel automatic matrix training framework to construct an appropriate substitution matrix for the alphabet. This training strategy did not need any information about known alignments, e.g. PALI [43], that most previous strategies have required. Using different training data and update rules, the self-training methodology can be applied to various alphabets. For example, instead of protein classifications, we could consider RMSD in the update rules to tune the matrix. In Table 12, we summarize the properties of the structural alphabets and design methods evaluated in this study.Table 12Summary of properties of structural alphabets and alphabet designsStructural AlphabetTung & Yangde Brevern et al.Our SAAlphabet Size231618How the alphabet size was determinedPrespecifiedIterative shrinkingBICClusteringk-meansSOM+HMMSOM+k-meansData SetPreprocessed (Pair Database)Preprocessed (PBE-SELECT)No preprocess (nrPDB)Substitution MatrixBLOSUM-likeBLOSUM-likeSelf-TrainingRequires known alignments to build matrixYesYesNoApplicabilityLimitedLimitedModular design More flexibleWe demonstrated that our pipeline could produce a biologically meaningful structural alphabet. We compared SA-FAST, a search tool based on FASTA combined with our alphabet and substitution matrix, with other search tools. The results showed that SA-FAST was very competitive in its predictive accuracy and alignment efficiency for database-scale searches. In addition, we compared our alphabet with others in difficult cases of pairwise alignment. The number of residues aligned and the RMSD superpositions indicated that our structural alphabet was not only comparable to other alphabets but also performed competitively with structural alignment tools.We found several advantages to using a 1D structural alphabet. First, 1D representations of protein structures are easier to compare and more economical to store. Second, previously designed and widely used 1D sequence alignment tools can be applied directly to protein structure and sequence analysis. Third, 1D-based approaches can serve as pre-processors to filter out irrelevant proteins prior to the application of more computationally intensive structural analysis tools.ConclusionThese results are encouraging and we can extend this work in several directions. Firstly, we can use more complete datasets for substitution matrix training to increase the sensitivity and selectivity of future database searches. Secondly, we can combine other alignment tools, in addition to FASTA, with our substitution matrix and evaluate the performance of these different combinations. Thirdly, to increase the performance of MEME in structural motif detection, we could modify MEME or develop a new motif-finding tool specifically for our structural alphabet. MEME was originally designed to find motifs in amino acid and nucleic acid sequences. Currently, we use MEME to detect protein motifs and we have demonstrated that it can recover some of the structural sub-domains described by our structural alphabet. Finally, several structural alphabets have been developed based on different protein structural characteristics. It would be worthwhile to conduct a thorough comparative study and evaluate the feasibility of combining different alphabets. The combination of complementary structural alphabets would increase their overall applicability and characterize 3D protein structures more completely.MethodsThe use of frequent local structural motifs embedded in a polypeptide backbone has recently been shown to improve protein structure prediction [1,22]. The success of this strategy has paved the way for further studies of structural alphabets and has enabled the application of standard 1D sequence alignment methods to 3D protein structural searches. In this study, we combined several computational methods into a new approach to the design of a protein structural alphabet. We then developed an automatic matrix training framework that could generate appropriate substitution matrices for new alphabets when applied in standard 1D sequence alignment methods, such as FASTA [27].Structural alphabet designWe used proteins from the nrPDB [44] in our study with the aim of building a structural alphabet suitable for all proteins. The same approach could easily be applied to other databanks as well. We transformed each protein backbone into a series of dihedral angles (ϕ and ψ, neglecting ω) [15,22]. Following de Brevern et al. [15], our analysis was limited to fragments of five residues because this number of residues is sufficient for describing a short α helix and a minimal β structure. Fixing the window size at five residues, we applied a sliding-window approach to each protein sequence in nrPDB and gathered 20,953,584 fragment vectors. Each protein fragment, associated with α-carbons Cα(i-2), Cα(i-1), Cα(i), Cα(i+1), and Cα(i+2), was represented by a vector of eight dihedral angles [ψi-2, ϕi-1, ψi-1, ϕi, ψi, ϕi+1, ψi+1, ϕi+2] Unlike previous works that directly applied SOMs to obtain clusters of backbone fragments as the basis of the structural alphabet [28], in our approach the SOM was only part of the process that determined the number of letters required for the alphabet. We did not build our alphabet directly from the clusters found by SOM.The U-matrix is one of the most widely used methods for visualizing the clustering results of the SOM. The U-matrix shows the distances between neighboring reference vectors and can be visualized efficiently using the greyscale [45]. We conducted a post-process on the U-matrix using a minimum spanning tree algorithm. Based on the grey levels in the U-matrix, all of the map units were linked in the minimum spanning tree. Given a threshold θ determined by BIC, we partitioned the entire tree into several disconnected subtrees by removing the links between map units with grey levels below θ.Let S = {si | i = 1...M} be the set of map units we wished to cluster. Each map unit si is associated with a collection of input data points, Xi={xji|j=1...ni}, mapped to the map unit si. Let Ck = {ci | i = 1...k} be the clustering of map units S with k clusters. We modeled each cluster ci as a multivariate Gaussian distribution N(μi, Σi), where μi and Σi were estimated as the sample mean and the sample covariance from Xi, respectively. The number of parameters for each cluster was thus d+12d(d+1), where d = 8 in our case. We defined BIC(Ck) as:BIC(Ck)=∑i=1k{−12nilog⁡|∑i|}−λ12(d+12d(d+1))klog⁡Nwhere N=∑i=1kni and λ, the penalty weight, was set to 1.We chose the threshold θ that maximized BIC(Ck). For example, for an SOM with 200 × 200 map units, the threshold θ that maximized BIC(Ck) was 21. The number of subtrees becomes the structural alphabet size. Because the SOM can be viewed as a topology preserving mapping from input space onto the 2D grid of map units, the number of map units can affect the clustering result. We systematically varied the number of units and repeated the above process. We selected the most frequent number of clusters as the alphabet size. After a series of systematic tests, we found that 18 was the most frequent number of clusters; therefore, 18 letters became size of our structural alphabet.Rather than adopt the two-level approach that first trains the SOM then performs clustering on the trained SOM after determining the alphabet size [28], we applied the k-means algorithm to the input data vectors directly to obtain the clusters. The SOM established a local order among the set of reference vectors such that the closeness between two reference vectors in the Rd space was dependent on how close the corresponding map units were in the 2D array. Nevertheless, an inductive bias of this kind might not be appropriate for structural alphabets since the local order does not always faithfully characterize the relationship between structural building blocks and can sometimes be misleading. For example, forcing the topology to preserve mapping from the input space of α-helix and β-strand to a 2D grid of units could be harmful to clustering. Therefore, we used the SOM only to visualize the alphabet size and relied on the k-means algorithm to extract the local features directly from the input data that actually reflected the characteristics of the clusters. The centroid of each cluster forms the prototypical representation of each alphabet letter. We performed k-means clustering 50 times, starting with different random seeds, all using k = 18. We computed the Euclidean distances from each fragment in each cluster to its centroid as the intra-cluster distance; we also calculated the centroid-to-centroid distance. We kept the clustering result that had the minimum ratio of the average intra-cluster distance to the centroid-to-centroid distance. Given this result as the basis for the structural alphabet, we could transform a protein into a series of alphabet letters by matching each of its fragments against our alphabet prototypes.Automatic substitution matrix trainingThe substitution matrix used to align proteins represented by structural alphabets affects the alignment accuracy. The matrix is a crucial factor in the success of applying a 1D sequence alignment tool to search for similar 3D structures. The simplest matrix that can be used is the identity matrix. Some authors have applied an HMM approach to define the matrix [33], while others have adopted approaches similar to the development of BLOSUM matrices [24,31,45]. The identity matrix ignores possible acceptable alphabet letter substitutions, significantly limiting its applicability. The BLOSUM-like approach requires alignments of homologous proteins before calculating the log-odd ratios as the entries in the matrix; however, reliably aligned protein blocks might not always be available for log-odd ratio estimation. To avoid these drawbacks, we trained the substitution matrix without using the known blocks of protein alignments. This matrix training procedure can be applied regardless of how the alphabet is derived.There are three components in the matrix training framework: an alignment tool with a substitution matrix, training data, and a matrix trainer. We used FASTA as the alignment tool and the non-redundant proteins in SCOP1.69 with sequence similarity less than 40%, excluding the families with less than five proteins and those proteins used for validation, as the training dataset. Note that the training dataset was only 9.62% of the entire SCOP1.69. The test data we used in the later experiments (see Results section) did not overlap with our training examples. We started by using the identity matrix as the initial substitution matrix where the score for a match was 1, and for a mismatch, 0. Each protein in the training dataset was iterated as a query for FASTA to search the rest of the dataset for similar proteins. If a protein returned by FASTA belonged to the same family as the query, we considered the case a positive hit; otherwise it was a negative hit. Those proteins not returned by FASTA but in the same family as the query were considered misses. We gathered the alignments of all positive hits and misses and computed the log-odd ratios to build the positive matrix based on the alignments. Similarly, we constructed the negative matrix using the alignments of negative hits, The matrix trainer updated the current substitution matrix S(t) to S(t+1) as follows:S(t+1) = S(t) + MM = [Wp·(P - S(t)) - Wn·(N - S(t))]·τWp = (|positive_hits| + |misses|)/|taining_data|Wn = |negative_hits|/|training_data|where P and N are the positive and the negative matrix, respectively, τ is the learning rate (similar to the learning rate in neural networks), and Wp and Wn are the weights. The weights were defined as the proportion of the total number of positive hits and misses to the training data size and the ratio of the number of negative hits to the training data size, respectively. We repeated the update process to train the substitution matrix until there were no changes in the matrix, that is, the number of both the positive and the negative hits remained constant. This converged matrix was the final substitution matrix that we combined with FASTA to become a new alignment tool named SA-FAST. SA-FAST was used to demonstrate the applicability of our new alphabet and matrix. The training framework appears in Figure 7.Figure 7System architecture of the matrix training framework.Authors' contributionsS–YK implemented the structural alphabet design pipeline and conducted the experiments. Y–JH designed the BIC procedure, the matrix training framework and experiments, and supervised this study. Both authors read and approved the final manuscript.\n\nREFERENCES:\nNo References"
+}

batch_11/PMC2529408.json

@@ -0,0 +1,4 @@
+{
+    "id": "PMC2529408",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2529408\nAUTHORS: Steven L. Salzberg, Daniel D. Sommer, Daniela Puiu, Vincent T. Lee\n\nABSTRACT:\nRecent improvements in technology have made DNA sequencing dramatically faster and more efficient than ever before. The new technologies produce highly accurate sequences, but one drawback is that the most efficient technology produces the shortest read lengths. Short-read sequencing has been applied successfully to resequence the human genome and those of other species but not to whole-genome sequencing of novel organisms. Here we describe the sequencing and assembly of a novel clinical isolate of Pseudomonas aeruginosa, strain PAb1, using very short read technology. From 8,627,900 reads, each 33 nucleotides in length, we assembled the genome into one scaffold of 76 ordered contiguous sequences containing 6,290,005 nucleotides, including one contig spanning 512,638 nucleotides, plus an additional 436 unordered contigs containing 416,897 nucleotides. Our method includes a novel gene-boosting algorithm that uses amino acid sequences from predicted proteins to build a better assembly. This study demonstrates the feasibility of very short read sequencing for the sequencing of bacterial genomes, particularly those for which a related species has been sequenced previously, and expands the potential application of this new technology to most known prokaryotic species.\n\nBODY:\nIntroductionGenome sequencing technology has moved into a new era with the introduction of extremely fast sequencing technologies that can produce over one billion base pairs (bp) of DNA in a single run. Some of the fastest methods today, based on strategies such as cyclic reversible termination [1] and ligation-based sequencing [2], produce the shortest read lengths, ranging from 15–50 bp. These lengths are sufficient for resequencing projects, including efforts to sample the human population, but they have yet to prove as useful for sequencing of novel species. The difficulty is that no existing assembly algorithms can accurately reconstruct a genome from such short reads [3].The first published report of a bacterial genome sequence from “short” reads used pyrosequencing technology, which was able to generate reads averaging 110 bp. That study [4] demonstrated the feasibility of assembling the small bacterial genome of Mycoplasma genitalium (580,069 bp) from reads that covered the genome 40-fold. This combination of coverage and read length allowed Margulies et al. to generate contiguous stretchs of DNA (contigs) averaging 22.4 kilobases (kb). Results using pyrosequencing have improved steadily as read lengths have increased to 250 bp and longer, but the difficulty of de novo assembly has raised questions about the utility of alternative sequencing technologies—those that produce reads shorter than 50 bp—for genome sequencing projects.Assembly of novel strains and species—where the genome has not previously been sequenced—from very short reads has proven more difficult, although simulation studies have indicated that it should be possible [5]. A recent study showed that a combination of pyrosequencing reads (average length 102 bp) and paired-end sequencing could be used to assemble a 4 million base pair (Mbp) genome into just 139 contigs, linked together in 22 scaffolds [6]. Another recent effort used a hybrid strategy that mixed pyrosequencing (110 bp reads) and traditional Sanger sequencing to produce draft assemblies of marine microbes [7]. In contrast, the very short reads generated by the Solexa Sequence Analyzer have thus far been useful primarily for polymorphism discovery in the human genome, for resequencing and polymorphism discovery in Caernohabditis elegans\n[8], and for other applications such as ChIP-seq [9], which identifies genomic regions bound by transcription factors.The very short reads—currently 30–35 bp—produced by CRT technologies such as Solexa present a far more difficult assembly problem. Standard assembly algorithms such as Arachne [10],[11] and Celera Assembler [12] cannot process such short reads at all, spurring the development of several new algorithms designed for short reads, including SSAKE [13], Velvet [14], Edena [15], and ALLPATHS [16]. These latter methods can handle Solexa data (though ALLPATHS has the additional requirement that the sequences must be paired-end reads), but they produce highly fragmented assemblies when provided with whole-genome data from a bacterial genome. The inherent problem with very short reads is that every repetitive sequence longer than the read length causes breaks in the assembly.To demonstrate the feasibility of assembling a bacterial genome from 33 bp reads, using related genomes to assist the process, we chose Pseudomonas aeruginosa strain PAb1, a highly virulent strain isolated from a frostbite patient. P. aeruginosa is a ubiquitous environmental bacteria of clinical importance as the leading cause of gram-negative nosocomial infections [17],[18]. Several P. aeruginosa genomes have been sequenced previously, including two laboratory strains: PAO1 (6,264,404 bp), originally isolated from a wound, and PA14 (6,537,648 bp) isolated from a burn [19],[20]. PA14 and PAO1 are ∼99% identical across the 6.05 Mbp shared by both genomes, and their similarity to PAb1 allowed us to improve the assembly and provided a means to check its accuracy. One of our goals in sequencing PAb1 was to identify genomic differences that contribute to its altered pathogenicity.Here we report the assembly of P. aeruginosa PAb1 entirely from 33 bp reads, using a novel assembly strategy that takes advantage of related genomes and homologous protein sequences. The assembly is of very high quality, comparable to or better than draft assemblies produced using earlier sequencing technologies. This study shows that a novel bacterial genome can be sequenced entirely with very short read technology, without the use of paired-end sequences (which are not available from some short-read sequencers), and assembled into a high-quality genome. Even at 40-fold coverage, the amount of sequence represents just one-quarter of a single sequencing run on a Solexa instrument, which brings the sequencing cost easily within the reach of most scientists. By making all of our assembly software free and open source, we hope to further bring down the barriers to desktop whole-genome sequencing.Algorithm for Assembly of Very Short ReadsWe generated 8,627,900 random shotgun reads from P. aeruginosa PAb1 using Solexa technology. All reads were exactly 33 bp in length.We used four distinct computational steps to assemble the genome of PAb1. For the initial step, we used the comparative assembly algorithm AMOScmp [21], which aligns all reads to a reference genome, and then builds contigs based on these alignments. The algorithm gains efficiency by avoiding the costly all-versus-all overlapping step, which is particularly difficult with very short reads due to the high incidence of false overlaps [13]. We modified AMOSCmp by tuning the MUMmer software [22], which is run within AMOScmp, to look for exact matches to the reference genome of at least 17 bp, allowing at most two mismatches in each read. We found that careful trimming of the reads based on their matches to the reference produced better assemblies than un-trimmed reads. The initial assembly used 7,500,501 reads, leaving 1,127,399 as singletons (Table 1). The PAb1 genome is closer to PA14 (99.4% identical for 92% of the PAb1 genome) than to PAO1 (99.0% identical for 90% of the PAb1 genome), and we therefore used PA14 as the primary reference for orienting the contigs.10.1371/journal.pcbi.1000186.t001Table 1Major steps in the assembly of P. aeruginosa from 33 bp Solexa reads.Assembly StepInputNumber of ContigsContigs >200 bpLargest ContigSingletonsAMOScmp with PA148,627,900 reads2,053428170,4851,127,399AMOScmp with PAO18,627,900 reads2,79786575,6261,592,525Merged comparative assemblies4,850 contigs1,850306236,4721,066,226Gene-boosted assembly306 contigs120120512,638NADe novo assembly by Velvet8,627,900 reads10,684738216,2391,241,079Merged gene-boosted and Velvet assemblies120 contigs, 7382 contigs7676512,638822,210The first column indicates the assembly strategies described in the text. Singletons refers to the number of reads that were not used to produce the contigs generated by each method.Our second step was a novel enhancement to the comparative assembly strategy, in which we used multiple reference genomes (Figure 1). We used the complete genomes of both PAO1 [19] and PA14 [20] separately to build multiple comparative assemblies, and found that PA14 produced the better assembly, comprising 2,053 contigs containing 6,206,284 bp. (We also used the PA7 strain, but its greater evolutionary distance made it less useful.) The bulk of the sequence was contained in 157 contigs longer than 10 Kbp, which collectively covered 5,568,616 bp. There were 331,364 bp in the PA14 genome that were not covered by the initial assembly, due to divergence between the two strains. However, the gaps in the comparative assembly based on PAO1 occurred in different locations due to differences between the strains. The best assembly based on PAO1 comprised 2797 contigs covering 6,043,652 bp.10.1371/journal.pcbi.1000186.g001Figure 1Comparative assembly using multiple genomes.The target genome is shown in the center, aligned to two related genomes, A and B. The DNA sequence of the target diverges from the reference genomes in distinct loci, labeled X, Y, and Z. The comparative assembly based on genome A contains a gap corresponding to region Y, while the assembly based on genome B contains two gaps, corresponding to X and Z. The merged assembly will cover all of the target genome with no gaps.We aligned the two assemblies to one another to identify locations where a contig in the PAO1-based assembly might span two or more contigs in the PA14-based assembly (Figure 1). For each such case, we filled the gap with the sequence from the PAO1 assembly using the Minimus assembler [23] to stitch together the contigs. This algorithm closed 203 gaps, reducing the number of contigs to 1850, of which all but 305 were <200 bp. The bulk of the genome, 5,949,162 bp, was contained in just 113 contigs of 10,000 bp or longer. Note that the overlapping contigs between the two assemblies did not agree perfectly. In order to produce a clean merged assembly, we re-mapped the reads to the contigs using AMOScmp to create consistent multi-alignments.The third step used a novel algorithm, gene-boosted assembly. For this step, we took the contigs from the previous step and identified protein-coding genes using our annotation pipeline, which is based on Glimmer [24] and Blast [25]. Because amino acid sequences are much more conserved than nucleotide sequences, we were able to use the predicted protein sequences (primarily but not exclusively from other Pseudomonas species) to fill gaps even where the DNA sequences diverged. The annotation pipeline identified 5,769 proteins in the 305 longest contigs.From the initial annotation, we identified those genes that extended beyond the ends of contigs or that spanned the gaps between contigs. We extracted the amino acid sequences corresponding to these gap positions, with a small buffer sequence included on each side of each gap. Next we used tblastn [25] to align each protein sequence to all the unused reads translated in all 6 frames (Figure 2). This step identified, for each gap, a small set of reads that would fill in the missing protein sequence, and the tblastn results provided initial locations for a multiple alignment. We then used a new program, ABBA (Assembly Boosted By Amino acids), to assemble the reads together with the flanking contigs and close the gaps. This gene-boosted assembly protocol extended many contigs and closed 185 gaps, ranging in length from 14–1095 bp, reducing the number of long contigs to 120.10.1371/journal.pcbi.1000186.g002Figure 2Gene-boosted assembly.All contigs are aligned with predicted gene sequences to identify genes that span 2 or more contigs. The DNA sequences of these spanning genes are cut out with a small buffer on each end. The amino acid translation of each gene fragment is then searched against a translated database of all singleton reads that have not yet been placed in the assembly. Finally, the reads identified by this process are assembled together with the two contigs to fill in the gap.As a separate test, we conducted a gene-boosted assembly of PAb1 using only the annotated proteins from PA14—without any reference genomic sequence. For this experiment, we aligned all the translated reads to each protein and used ABBA to assemble each one. For 4,572 of the proteins, ABBA produced a single contig that covered the entire reference protein, and another 831 proteins assembled into a few contigs. Thus 5,403 out of 5,602 (96%) of the PAb1 proteins can be assembled using a pure gene-boosting approach, and additional proteins would likely be assembled if we used a large set of proteins for boosting. This demonstrates that in the absence of a closely related genome sequence, gene-boosted assembly can use protein sequences—which diverge much more slowly than genomic DNA—to assemble most of the genes of a new bacterial strain, although the results will lack global genome structure information.The fourth step of our method identified any remaining DNA sequences that were (a) unique to PAb1 and (b) outside predicted gene regions. We separately constructed pure de novo assemblies of the 8.6 million Solexa reads using SSAKE, Edena, and Velvet. The Velvet assembly was the best of the three, creating 10,684 contigs, the longest being 16,239 bp (Table 1). We used MUMmer to align these contigs to the 120 long contigs in our scaffold from the previous step, and identified cases where de novo contigs spanned gaps or extended contigs. This step allowed us to close 46 gaps, reducing the number of contigs in our main scaffold to 74. After removing Velvet contigs that were already contained in our scaffold, we had 436 unplaced de novo contigs spanning 416,897 bp. The longest unplaced contig was 10,493 bp.Results/DiscussionOur final assembly contains one large scaffold with 76 contigs whose total length is 6,290,005 bp, with the longest contig at 512,638 bp. The 436 unplaced contigs, which should fit into the remaining gaps, represent sequence that is unique to PAb1. Our annotation shows that most of these contigs contain genes that are homologous to other Pseudomonas species. Several contigs contain bacteriophage genes, pointing to recent phage insertion events in PAb1. The final assembly thus consists of 512 contigs covering 6,706,902 bp, with 94% of the bases in a single large scaffold. Approximately 9% of the reads were not used in the assembly (Table 1); many of these can be mapped to contigs if we use relaxed matching criteria, indicating that they represent low-quality data. Our annotation of the PAb1 genome identified 5,602 protein-coding genes, as compared to 5,568 for PAO1 and 5,892 for PA14.All Solexa reads have been deposited in the Short Read Archive at NCBI, and the final genome sequence and annotation have been deposited in GenBank as sccession ABKZ01000000.We have demonstrated that it is possible to sequence and assemble a bacterial genome from deep sequencing using 33 bp reads. The final assembly has 40.3× coverage, with very high agreement among the individual reads at the vast majority of positions in the genome. To measure the accuracy of individual reads, we examined all positions in the assembly with >20× coverage, which yielded 5.9 million positions. If we count as errors any bases that disagree with the consensus at those positions, we get an estimate based on internal consistency that the error rate per read is 1.04%. Based on this estimate, the expected number of errors for regions of the genome with coverage of >20× is close to zero, except for systematic errors such as difficult-to-sequence regions. This illustrates how the great depth of sequencing possible with short-read technology produces higher quality assemblies—in regions with deep coverage—than would conventional Sanger sequencing at a typical 8× coverage depth.We evaluated the coverage to determine if the Solexa sequences were biased towards any portion of the genome, and found a small bias towards high-GC regions, which comprise most of the genome. In particular, regions with 60–70% GC, which comprised 79% of the genome, had 40× coverage. In contrast, regions with 50–55% GC (1.5% of the genome) had 14× coverage, and regions with <50% GC (1.1% of the genome) had just 5× coverage.The alignment of P. aeruginosa PAb1 to strain PA14, which matches at 99.4% identity for >90% of the genome, can be used to provide an estimate of the sequencing accuracy. To assess the question of whether differences between our assembly and the PA14 genome represented true differences or sequencing errors, we aligned the two genomes and identified all single nucleotide polymorphisms (SNPs). Out of 5,568,550 aligned bases from the longer PAb1 contigs, 5,537,508 agreed with PA14 and can be presumed correct. For each of the remaining 31,042 SNPs, we examined all reads that were assembled at that point and assessed whether (a) the depth of coverage was adequate, and (b) the PAb1 reads agreed on the consensus base. The coverage was 10-fold or greater for 95% of these SNPs. Using the conservative assumption that a SNP might be in error if the inter-read agreement was less than 80%, we found 1157 positions (out of 5,568,550) that might be sequencing errors. We also found 1104 insertions and deletions (indels) in the aligned regions, and our assembled reads were in perfect agreement for 917 of these. If we assume conservatively that the other 187 indels are errors, then considering both SNPs and indels, the accuracy of the assembled genome is greater than 99.97%.The assembly is sufficiently complete that we can confidently infer that genes are missing if their expected positions fall in the midst of contigs. Although deeper analysis will be presented in a followup paper, we note that the PAb1 strain is known for its hypermotility on low percentage agar media. Our sequence contains most of the genes required for swimming motility in P. aeruginosa\n[26], but is missing part of the pathway used by cyclic-di-GMP, a secondary signaling molecule, that has been implicated in repressing swimming motility [27],[28]. By searching all of the known P. aeruginosa genes in this pathway [29],[30],[31], we found that three genes encoding diguanylate cylase and phosphodiesterase are missing: PA2771 and PA2818 (arr) from the PAO1 strain, and PA14_59790 (pvrR) from the PA14 strain [32],[33]. All three of these genes are located in chromosomal regions previously indicated as hyper-variable based on genomic hybridizations [29]. The altered gene content of PAb1 in the regulatory pathways repressing flagella may contribute to its observed hypermotility.The new algorithm described here make it possible for any scientist to acquire the entire genome of a bacterium at high speed and very low cost. One limitation of our method is that it depends on the existence of related genomes (for the comparative assembly step) and protein sequences (for the gene boosting step). However, GenBank already contains the complete genome sequences for >650 microbial genomes, and draft sequences for nearly 1000 more. For many of these species, much larger numbers of related strains and species have yet to be sequenced. Our method opens the door to the use of whole-genome sequencing to study entire collections of bacteria, to rapidly identify genotypes from mutagenized genetic screens, and for other analyses that were previously too costly or technically infeasible. The gene-boosted assembly technique applies equally well to both short and long-read sequencing methods, and should also work for assembling the gene-containing regions of much larger genomes.MethodsGenomic DNA was extracted by SDS lysis, proteinase K digest, and phenol/chloroform extraction. Sequencing was performed by Illumina using the 1G Genome Analyzer, also known as the Solexa sequencer. The 8.6 million reads represent 1/4 of the current capacity of a flow cell. For sequencing trimming in step 1, we mapped all reads to the initial assembly and then trimmed up to three bases from the 3′ end when those bases failed to match a contig. The AMOScmp pipeline for trimming and short read assembly is described at http://cbcb.umd.edu/research/SR-assembly.shtml. Contig merging in step 2 of our algorithm used the merger program from the EMBOSS package [34]. The Edena, Velvet, and ssake assemblers were run with a wide range of parameters in order to optimize them for the data used in this study, with the best results coming from Velvet with a minimum overlap requirement of 24 bases. (The other methods created more numerous, shorter contigs.) The ABBA assembler has been added to the free, open-source AMOS assembler package, which also includes the AMOScmp assembler. ABBA can be found at http://amos.sourceforge.net/docs/pipeline/abba.html. AMOS and additional modules developed in this study are freely available from http://cbcb.umd.edu/software, and the MUMmer system is freely available at http://mummer.sourceforge.net.\n\nREFERENCES:\n1. MetzkerML\n2005\nEmerging technologies in DNA sequencing.\nGenome Res\n15\n1767\n1776\n16339375\n2. MardisER\n2008\nThe impact of next-generation sequencing technology on genetics.\nTrends Genet\n24\n133\n141\n18262675\n3. ChaissonMJPevznerPA\n2008\nShort read fragment assembly of bacterial genomes.\nGenome Res\n18\n324\n330\n18083777\n4. MarguliesMEgholmMAltmanWEAttiyaSBaderJS\n2005\nGenome sequencing in microfabricated high-density picolitre reactors.\nNature\n437\n376\n380\n16056220\n5. WhitefordNHaslamNWeberGPrugel-BennettAEssexJW\n2005\nAn analysis of the feasibility of short read sequencing.\nNucleic Acids Res\n33\ne171\n16275781\n6. 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+}

batch_11/PMC2529470.json

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+{
+    "id": "PMC2529470",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2529470\nAUTHORS: Jing Zhao, Atsumi Iida, Yasuo Ouchi, Shinya Satoh, Sumiko Watanabe\n\nABSTRACT:\nPurposeGlycoprotein m6a (M6a) is a cell-surface glycoprotein that belongs to the myelin proteolipid protein family. M6a is expressed mainly in the nervous system, and its expression and function in mammalian retina have not been described. Using proteomics analysis of mouse retinal membrane fractions, we identified M6a as a retinal membrane protein that is strongly expressed at embryonic stages. Our aim was to reveal the function of M6a in development of mouse retina in this work.MethodsDetailed expression pattern of M6a was examined by immunostaining using frozen sections of mouse retina obtained at various developmental stages. For functional analysis of M6a in mouse retinal development, we performed retorovirus-mediated overexpression of M6a in mouse retinal explant culture. Then, cell differentiation, proliferation and structural maturation of the cells were examined.ResultsM6a transcripts were strongly expressed in embryonic retina. After completion of retinal differentiation, the level of expression decreased as mouse development progressed. Immunohistochemistry showed that in the immature mouse retina, M6a was strongly expressed in the axons of retinal ganglion cells. After birth, M6a expression was confined to the inner plexiform layer, and finally, to the inner and outer plexiform layers of adult mouse retina. M6a expression was completely paralleled by that of the synaptic marker, synaptophysin. Mouse retinal progenitor cells that overexpressed M6a following retrovirus-mediated gene transfer were subjected to in vitro explant or monolayer cultures. The neurite outgrowth of M6a-overexpressing retinal cells was strikingly enhanced, although M6a did not affect differentiation and proliferation.ConclusionsThese results suggest that M6a plays a role in retinal development by regulating neurites, and it may also function to modulate synaptic activities in the adult retina.\n\nBODY:\nIntroductionThe mature neural retina is organized into a three-layered structure consisting of Müller glia, astrocytes, and six types of neurons. These cells are assumed to differentiate in a precise histogenic order from a single population of multipotent retinal precursors [1]. Various molecules, such as transcription factors and neurotrophic factors, have been reported to play important roles in retinal cell differentiation [2]. However, the intrinsic properties of retinal cells at different developmental stages are still vague. This is in part due to the lack of markers that can identify distinct stages of retinal progenitor cells. In our previous studies, we have tried to identify markers of retinal progenitor cells by employing flow cytometry and cell sorting. Using a panel of antibodies against cell-surface antigens, we screened mouse retinal cells at various developmental stages for reactivity. This technique obtained unique expression patterns for more than 30 antigens in the developing retina. Among them, some CD antigens, such as SSEA-1 (CD15) and c-kit (CD117) were identified as retinal progenitor cell markers in early and late immature stages, respectively [3,4].Since this approach only identifies known molecules, we used proteomics to examine the comprehensive expression profile of total membrane proteins from embryonic and adult mouse retina. Information about membrane proteins, which are expressed in a specific manner in the developing retina, may not only serve as a tool for purification of retinal subfractions by cell sorting, but may also be useful for analyzing the regulation of retinal development by receptor-signaling and cell surface molecules. To establish such a database, we used shotgun analysis and a nanoflow liquid chromatography-mass spectrometry/ mass spectrometry (LC-MS/MS) system to examine total protein expression in purified membrane fractions [5]. We identified several membrane-associated proteins which are expressed in embryonic retina [5], and among the proteins, we focused on glycoprotein m6a (M6a) in this work.M6a is a transmembrane protein that belongs to the myelin proteolipid protein (PLP) family. The M6a gene encodes a 278 amino acid protein that contains four putative transmembrane domains with both the N- and C-termini facing the cytosol. PLP constitutes the most abundant protein (approximately 50%) in the central nervous system (CNS) myelin sheath and is involved in signaling through integrins in oligodendrocytes [6]. Although PLP and its splice variant DM20 are glial proteins, M6a is found exclusively in neurons [7]. M6a is present on the postmitotic neurons of the developing neural tube at embryonic day 9 (E9) and is continuously expressed in multiple regions of the CNS in the mouse. Furthermore, the M6a protein is located at the leading edge of the growth cone in cultured cerebellar neurons [8]. Recent studies have suggested the importance of M6a in the process of neural development, such as neurite extension, survival [9], and differentiation [10]. Furthermore, M6a has been found to play an important role in neurite outgrowth and filopodium and spine formation, and may also be involved in synapse formation in cultured hippocampal cells [11]. These findings indicate the possible involvement of M6a in neuronal survival and differentiation. However, the expression pattern and function of M6a in the mouse retina have not been investigated to date.We identify M6a as a gene that is expressed in the embryonic retina and reveal the expression patterns of M6a in the neural processes, including the nerve fiber layer (NFL), inner plexiform layer (IPL), and outer plexiform layer (OPL), of the immature mouse retina. We also show that the expression of M6a parallels that of synaptophysin. Forced expression of M6a in mouse retinal explant cultures resulted in enhancement of neurite extension, which suggests that M6a plays important roles in the regulation of neurites in the embryonic retina.MethodsIsolation of retina from miceICR mice were obtained from Japan SLC, Inc. (Hamamatsu, Japan) and Clea Japan, Inc. (Tokyo, Japan). Mice are housed under 12/12 light/dark cycles in standard shoebox cages with water and food at 23°C. The day that a vaginal plug was observed was considered to be embryonic day 0 (E0), and the day of birth was marked as postnatal day 0 (P0). All animal experiments were approved by the Animal Care Committee of the Institute of Medical Science, University of Tokyo. Mice were euthanized by decapitation or cervical dislocation under anesthesia.Plasmid construction and production of retrovirusThe mouse M6a cDNA was cloned by RT–PCR from pooled mouse cDNA from P1 retina. The primers were designed based on the sequences available in the database. A full-length fragment of M6a was cloned into the Not I site of the pMXc-IRES-EGFP retrovirus vector (kindly provided by Dr. T. Kitamura, University of Tokyo, Japan), which directs expression of the cloned genes together with enhanced green fluorescent protein (EGFP) from upstream long terminal repeat (LTR) promoter. Production of the retrovirus was performed using the PLAT-E packaging cell line [12] as previously described [13]. Briefly, PLAT-E was transfected with retrovirus vectors containing various genes by using Fugene6 transfection reagent (Roche, Indianapolis, IN) according to the manufacturer’s instructions. Two days after transfection, cell supernatants containing retrovirus were harvested and concentrated by centrifugation in a centrifugal filter device (Millipore, Billerica, MA).RT–PCRTotal RNA was purified from mouse retinas by use of Trizol reagent (Gibco BRL, Carlsbad, CA), and cDNA was synthesized with Superscript II (Gibco BRL). The primer sets were tested for several different cycling numbers by using rTaq (Takara, Otsu, Japan), and the semiquantitative cycle number was determined for each primer set. Bands were visualized with ethidium bromide.Retinal explant culture, retrovirus infection, and monolayer cultureRetinal explants were prepared as previously described [13]. Briefly, the neural retina was isolated on a Millicell chamber filter and placed with the ganglion cell layer facing upwards. The filters were inserted into six-well plates and cultured in 1 ml of explant culture medium (50% MEM with Hepes, 25% Hank’s solution, 25% heat-inactivated horse serum, 200 mM L-glutamine and 5,75 mg/ml glucose) [13]. Infection of retrovirus was done by exposing the concentrated virus solution to the explant for initial two days, as described previously [14]. Cells were then fixed with 4% paraformaldehyde (PFA) and frozen sectioned. For neurite extension assay, monolayer culture of retina was conducted. Retinal explants were prepared from E17 retinas and infected with retroviruses. After three days in culture, the cells were dissociated by treatment with 0.25% trypsin, and replated on eight-well chamber slides (BD Falcon, Bedford, MA) that were coated with ornithine (Sigma, St. Louis, MO) and fibronrctin (Sigma). The cells were cultured for an additional 11 days in Dulbecco’s modified Eagle’s medium/F-12 medium (Gibco BRL) that was supplemented with 1% fetal bovine serum (JRH Biosciences, Lenexa, KS) and 1% N2 (Gibco). Cells were fixed with 4% PFA and immunostained anti-green fluorescent protein (GFP; Clontech Laboratories, Palo Alto, CA) and anti-glutamine synthetase (GS; Chemicon, Temecula, CA) antibodies. The neurite lengths of GFP-positive and GS-negative cells were examined using Axioplan2 fluorescent microscopy (Carl Zeiss, Oberkochen, Germany). Then, neurite lengths of the cells were measured from randomly taken images using AxioVision 4.6 software (Carl Zeiss, Oberkochen, Germany) and Adobe Photoshop Elements 2 (Adobe Systems, San. Jose, CA). For reaggregation culture, retroviruses were infected into retinal explant cultures prepared from E17.5 retina. After overnight culture, cell were dissociated by treatment with 0.25% trypsin and used as donor cells. They were then mixed with three times number of dissociated retinal cells that had been isolated from the same brood and cultured overnight without virus infection. Then, the aggregates were cultured for eight days, and neurite extension was evaluated after immunostaining with anti-GFP antibody.Immunohistochemistry and antibodiesImmunohistochemistry of retinal explants was performed as previously described [4,13]. Briefly, frozen-sections of retinal explant were pre-incubated in a blocking solution containing 2% bovine serum albumin and incubated with the appropriate primary antibody solutions. The primary antibodies and their concentration in reaction solution used were as follows: 1:5,000 dilution anti-GFP polyclonal antibody (Clontech Laboratories), 1:1,000 anti-M6a (clone 321; MBL, Nagoya, Japan), 1:100 anti-Rho4D2 (kindly provided by Dr. R. S. Molday, The University of British Columbia, Vancouver, Canada), 1:1,000 GS (Chemicon), 1:500 anti-Hu C/D (Molecular Probes, Inc., Eugene, OR), 1:100 anti-protein kinase C (PKC; Oncogene Research Product, Boston, MA), and 1:100 anti-Ki67 (BD Biosciences) monoclonal antibodies. The primary antibodies were visualized by using appropriate second antibodies conjugated with 1:1,000 Alexa Fluor 488 or 546. All samples were sealed using VectaShield mounting media (Vector Laboratories, Burlingame, CA) containing DAPI for nuclear staining.BrdU labeling and detectionThree days after retrovirus infection, retinal explants were incubated with 5 μM bromodeoxyuridine (BrdU) for 24 h before they were harvested and fixed with 4% PFA. The samples were embedded in optimal cutting temperature (OCT) compound and frozen-sectioned. The sections were treated with 1 U/μl of DNase (Takara) in PBS for 1 h at 37 °C, and the incorporated BrdU was visualized immunohistochemically using an anti-BrdU monoclonal antibody (Roche, Indianapolis, IN) and the appropriate secondary antibodies.ResultsM6a is expressed in the neuronal processes of the mouse retinaTo obtain comprehensive expression profiles of the membrane proteins of embryonic and adult mouse retinas, we analyzed the membrane fractions for total proteins using shotgun analysis on a nanoflow LC-MS/MS system [5]. With this approach, we detected M6a in samples prepared from embryonic retinas, but not in samples from adult retinas [5]. M6a is known to be widely expressed in brain [8], whereas its detailed expression in the neural retinas of mammals has not been reported. We examined the expression of M6a mRNA over time, using semiquantitative RT–PCR (Figure 1A). M6a was expressed in E14 mouse retinas, and expression continued after birth with a slight decrease in the intensities of the bands between P12 and P15. Finally, a weak band was observed in the adult retina samples. We used immunohistochemistry to investigate the spatial and temporal expression patterns of M6a in mouse retina sections from various developmental stages.Figure 1Expression of M6a in mouse retinas from various developmental stages. A: Semiquantitative RT–PCR for M6a using total RNA extracted from mouse retinas at various developmental stages. Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was used as the control. B-E: Immunostaining of M6a in frozen sections of mouse retina from various developmental stages. Coimmunostaining was performed with anti-M6a and anti-βIII-tubulin (C), anti-glutamine synthetase (D), or antisynaptophysin (E) antibodies. The right two columns are magnified figure of the square area indicated by broken lines in left two columns B. The scale bar represents 100 μm. The following abbreviations are used in this figure: inner plexiform layer (IPL); outer plexiform layer (OPL); ganglion cell layer (GCL); neuroblastic layer (NBL); inner nuclear layer (INL); outer nuclear layer (ONL); retinal pigment epithelium (RPE).In the E14 retina, M6a was mainly and strongly expressed in the NFL, which consists of the axons of ganglion cells, but was not observed in the neuroblastic layer (NBL), which consists of proliferating retinal progenitor cells (Figure 1B). However, in P1 (Figure 1B,C) and P5 (data not shown) retinas, strong expression of M6a was confined to the IPL and also in NFL, which consists of innermost region, but not in ganglion cell layer (GCL). At P10 (data not shown) and in the adult retina, strong expression of M6a was detected in the IPL, and weak signals were observed in NFL, OPL, and inner nuclear layer (INL; Figure 1D).M6a protein colocalizes with synaptic markers of postmitotic cellsThe βIII tubulin protein is expressed at early stages by differentiated neurons, including ganglion and amacrine cells and by most retinal neurons up to P7 [15]. Double staining of retinal cells with anti-βIII tubulin and anti-M6a antibodies revealed that most cells were double-positive at these stages, which indicates that M6a is expressed on postmitotic mature neurons (Figure 1C). These results are comparable with a previous report that immunolabeling with anti-M6a antibodies was evident throughout the CNS of the embryonic mouse, but was absent from the zones of cell proliferation adjacent to the ventricles [8]. Not only for neurons, but M6a was also weakly expressed in processes of Müller glia cells, which are evident from the co-expression of M6a with Müller glia marker, glutamine synthetase (Figure 1D).Since previous reports have located M6a immunoreactivity in the synapses of the rat cerebellum and in the axon terminals of the rat cerebellar molecular layer [16], we examined the coexpression of M6a and the presynaptic marker, synaptophysin, by immunostaining with both anti-M6a and antisynaptophysin antibodies (Figure 1E). We found that M6a expression colocalized with that of synaptophysin from the embryonic to adult stages (Figure 1E).M6a overexpression does not affect cell fate and subretinal localization of retinal precursorsTo examine the biologic significance of M6a for retinal development, we investigated the effects of ectopically expressed M6a in a mouse retinal explant culture prepared from E17, which provides an excellent model to monitor retinal differentiation in vitro [13]. By E17, ganglion cells and a few other cell types have begun to differentiate, and after two weeks in culture, all of the retinal subpopulations have differentiated normally. The mouse retinal explant prepared from E17 was infected with retroviruses that encode either M6a-IRES-EGFP or IRES-EGFP (control) and cultured for two weeks. Since the retrovirus infects only mitotic cells, retinal precursor cells were assumed to be the major targets of gene transfer. We examined the subretinal localization of virus-infected cells. The number of M6a-EGFP-expressing cells was lower in the ONL and slightly higher in the INL than that of control EGFP-expressing retinal cells, but both differences were not statistically significant (Figure 2A). We then examined the differentiation of virus-infected cells by immunostaining frozen sections with antibodies against various marker proteins for retinal cell subpopulations (Figure 2B). The antibodies used were antirhodopsin for rod photoreceptors, anti-HuC/D for retinal ganglion cells, and amacrine cells, anti-PKC for bipolar cells, and anti-GS for Müller glia. The percentage of rhodopsin positive M6a-overexpressing cells was not significantly, but few times higher than that of control cells (Figure 2B). The other retinal cell populations were not affected by M6a overexpression, which suggests that M6a does not play a role in retinal differentiation.Figure 2Effects of forced expression of M6a on retinal differentiation and proliferation. A: Sublayer distributions of virus-transduced enhanced green fluorescent protein (EGFP)-positive cells in retinal explants. Retinal explants were infected with retorovirus particles that encode M6a and EGFP. After 14 days, the explants were harvested and frozen-sections prepared. Immunostaining was performed using an anti-EGFP antibody. The percentages of cells in each sublayer are shown. More than 200 cells were counted for each sample, and the standard deviation (SD) was calculated from three independent experiments. B: Differentiation of virus-infected cells examined by immunostaining to identify subpopulations within the retina. The percentages of marker-positive cells in the EGFP-positive population are shown. Rhodopsin for rod, HuC/HuD for amacrine, glutamine synthetase for Müller glia, and protein kinase C for bipolar were used as markers. More than 100 cells were examined for each sample, and the average value from three independent experiments is shown with the SD. C,D: Proliferation of M6a-expressing retinal cells was examined by measuring incorporation of bromodeoxyuridine (BrdU; C) or expression of the Ki67 antigen (D). BrdU was present for the final 24 h of four days of culture of retinal explants, and frozen sections were immunostained using antibodies against BrdU. The same samples were immunostained with the anti-Ki67antibody. The percentage of positive cells with SD are shown. Listed below each panel is the stage when each retinal explant was prepared and its culture period. The following abbreviations are in effect: outer nuclear layer (ONL); inner nuclear layer (INL); ganglion cell layer (GCL).M6a does not affect retinal precursor cell proliferationWe next examined whether M6a affects the proliferation of retinal cells by measuring BrdU incorporation. Retrovirus-mediated gene transfer into retinal explants prepared from E15.5 or E17.5 was conducted, and proliferating cells were labeled with 5 μM BrdU for the final 24 h of four days of culture. In all cases BrdU incorporation was slightly higher in the M6a-expressing cells than in control cells. Despite this general trend, statistical analysis revealed that the differences were not statistically significant (Figure 2C). Immunostaining the sections with the antiproliferation antigen, Ki67 antibody [17], produced slightly more Ki67-positive cells among the M6a-expressing cells than the control cells. These differences were not considered statistically significant, thus confirming the BrdU results (Figure 2D). Taken together, these results suggested a minor trend of increased proliferation associated with M6a but demonstrated that M6a does not regulate retinal cell proliferation significantly.To confirm the proliferation results, we performed a clonal assay [4] to test the proliferation capabilities of the virus-transfected M6a cells (data not shown). The colony sizes showed no significant differences between the control and M6a-transfected retinal cells.M6a overexpression promotes neurite outgrowth in retinaSince M6a has been implicated in neurite extension in brain, we investigated whether M6a was also involved in neurite extension in the retina, using monolayer cultures of retinas infected with retroviruses that encode control EGFP or M6a-IRES-EGFP. Neurite extension in monolayer cultures was examined by measuring the length of neurites from photographs taken under a fluorescence microscope. To distinguish neural cells from glial cells, we stained the retinal cells with antibody against GS, which is a glial cell marker, and evaluated the neurite lengths of the GS-negative and EGFP-positive cells. In both the control and M6a-expressing samples, approximately 60% of the cells extended neurites by 0–10 μm of neurite. However, when we compared the cell population distribution categorized by neurite length, we discovered that M6a-expressing cells extended longer neurites than did control virus-infected cells (Figure 3A). The average neurite lengths were greater than 10 μm for the M6a-overexpressing cells (39.2 μm) and control cells (25.5 μm; Figure 3B). We next confirmed these results with a different method of culture. We prepared reaggregation cultures by mixing dissociated virus-infected retinal explants prepared from E17.5 and dissociated retinal cells isolated from the same brood. Eight days later, percentage of M6a-overexpressing retinal cells bearing neurite over 30 μm length was about twice-times higher than that of control EGFP expressing cells (Figure 3C,D). This indicates that M6a plays a role in promoting the neurite extension during retinal development.Figure 3Effects of forced expression of M6a on retinal neurite extension. A,B: Neurite extension of virus-transduced enhanced green fluorescent protein (EGFP)-positive cells in retinal monolayer cultures. Retinal explants were infected with retorovirus particles that encode M6a and EGFP, and the cells were dissociated and subjected to monolayer culturing. After 11 days of culture, the cultures were harvested and stained with anti-GFP antibody. Neurite length was examined by measuring the EGFP-positive neurites on photographs taken under a fluorescence microscope. The length distribution percentages for the neurites are shown in A. The average lengths of neurites longer than 10 μm are shown in B. More than 100 cells were counted for each sample, and essentially the same results were obtained in three independent experiments. C,D: Neurite extension of virus-transduced EGFP-positive cells in retinal reaggregation cultures. Morphology of the M6a-EGFP virus-transfected retinal cells in reaggregation culture (C). The percentage of cells with neurite extensions of 30-100 μm or 100-200 μm in the M6a and control EGFP expressed retinal cells (D). Asterisk is p<0.05 by the Student's t-test.DiscussionM6a protein is targeted to the neuron processes of the murine retinaIn the present study, strong expression of M6a was detected in the ganglion cell axons and processes of cells located in the INL of the immature murine retina. A previous report on in situ hybridization of M6a in the Xenopus eye demonstrated expression of M6a mRNA in the INL and GCL, and weak expression in the ONL [18], which suggests that the expression of M6a in the retina is conserved in vertebrates.In mouse hippocampal tissues, M6a mRNA has been found to be expressed in granule cells of the dentate gyrus and pyramidal neurons in CA1 and CA3; immunoreactivity for M6a was concentrated in the regions of relatively dense synaptic contact [11]. Similar results have been obtained for the rat [16]. This implies that the translated M6a protein is targeted to processes distal to the somata. The expression of M6a paralleled that of the presynaptic protein, synaptophysin, in the mouse retina, suggesting that the subcellular distribution of M6a is the same in the brain and retina. It has been established that synapse formation by dissociated neurons in culture strongly correlates with focal accumulations of structures that can be labeled with antibodies against synaptic vesicle proteins, such as synaptophysin [19]. Therefore, our results indicate a possible role for M6a in the formation of synapses in neural retinas.M6a does not affect retinal progenitor cell differentiation and proliferationWe found that cell fate and subretinal localization of retinal progenitor cells were not affected by M6a overexpression. To date, there has been no report on the promotion of proliferation by M6a. On the other hand, it has been reported that the addition of anti-M6a antibody decreases the survival of dissociated neurons in culture and inhibits the extension of neurites in cultured cerebellar explants [9]. M6a belongs to the proteolipid protein (PLP)/DM20 family of myelin proteins. PLPs play a pivotal role in early oligodendrocyte differentiation and survival [20]. PLP members have been observed to form a complex with integrins and may participate in integrin receptor signaling in oligodendrocytes [21]. Given the high degree of homology between M6a and PLP, it is possible that this transmembrane protein is also related to integrin signaling. Our preliminary results from examining integrin family proteins in the developing retina reveal the expression of integrin αv in the embryonic mouse retina (unpublished results) which suggests that M6a plays role in retinal development through interaction with integrins to mediate signals for retinal development.M6a promotes neurite outgrowth in the retinaIn the present study, we observed promotion of retinal neurite extension by M6a. This protein has been reported to enhance neurite extension in rat pheochromocytoma PC12 cells and to induce an increase in the intracellular Ca2+ concentration of PC12 cells [10]. The anti-M6 antibody efficiently interfered with Ca2+ influx, which suggests that M6a acts as a Ca2+ channel blocker in PC12 cells. As a second messenger, Ca2+ has been shown to participate in neurite extension, filopodium and spine activity, and neuronal differentiation [22]. M6a possesses two PKC phosphorylation sites. Treatment of PC12 cells with a PKC inhibitor eliminates the ability of M6a to promote nerve growth factor-primed neurite extension [10]. Thus the putative phosphorylation sites in the cytosolic domains may facilitate M6a regulation and may be relevant for intracellular signaling. Therefore, the promotion of neurite extension by M6a in retinal cells seen in the present study is possibly associated with Ca2+ influx, and phosphorylation of PKC may remove this function. Future studies will focus on this hypothesis.\n\nREFERENCES:\nNo References"
+}

batch_11/PMC2530871.json

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+{
+    "id": "PMC2530871",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2530871\nAUTHORS: Shawn W Jones, Carlos J Paredes, Bryan Tracy, Nathan Cheng, Ryan Sillers, Ryan S Senger, Eleftherios T Papoutsakis\n\nABSTRACT:\nA detailed microarray analysis of transcription during sporulation of the strict anaerobe and endospore former Clostridium acetobutylicum is presented.\n\nBODY:\nBackgroundClostridia are of major importance to human and animal health and physiology, cellulose degradation, bioremediation, and for the production of biofuels and chemicals from renewable resources [1]. These obligate anaerobic, Gram-positive, endospore-forming firmicutes include several major human and animal pathogens, such as C. botulinum, C. perfringens, C. difficile, and C. tetani, the cellulolytic C. thermocellum and C. phytofermentans, several ethanologenic [2], and many solventogenic (butanol, acetone and ethanol) species [3]. Their sporulation/differentiation program is critical for understanding important cellular functions or programs, yet it remains largely unknown. We have recently examined the similarity of the clostridia and bacilli sporulation programs using information from sequenced clostridial genomes [1]. We concluded that, based on genomic information alone, the two programs are substantially different, reflecting the different evolutionary age and roles of these two genera. We have also argued that C. acetobutylicum is a good model organism for all clostridia [1]. Transcriptional or functional genomic information is, however, necessary for detailing these differences and for understanding clostridial differentiation and physiology. Key issues awaiting resolution include: the identification of the mid to late sigma and sporulation factors and their regulons; the orchestration and timing of their action; the set of genes employed by the cells in the mid and late stages of spore maturation; identification of candidate histidine kinases that might be capable of phosphorylating the master regulator (Spo0A) of sporulation; and some functional assessment of the roles of several sigma factors of unknown function encoded by the C. acetobutylicum genome. Furthermore, an understanding of the transcriptional basis of the complex physiology of this organism will go a long way to improve our ability to metabolically engineer, for practical applications, its complex sporulation and metabolic programs. Such information generates tremendous new opportunities for further exploration of this complex anaerobe and its clostridial relatives, and constitutes a firm basis for future detailed genetic and functional studies.Using a limited in scope and resolution transcriptional study, we have previously shown that it is possible to use DNA-microarray-based transcriptional analysis to generate valuable functional information related to stress response [4,5], initiation of sporulation [6] and the early sporulation program of C. acetobutylicum [7]. In order to be able to accurately study the transcriptional orchestration underlying the complete sporulation program of the cells, it was necessary to develop a more sensitive and accurate microarray platform, a better mRNA isolation protocol (in order to isolate RNA from the mid and late stationary phases), as well as to use a much higher frequency of observation and sampling. We also aimed to employ more sophisticated bioinformatic tools in order to globally interrogate any desirable cellular program and relate it to the characteristic phenotypic metabolism and sporulation of this organism. The results of this extensive study are presented here as a single, undivided story, which offers unprecedented insights and a tremendous wealth of information for further explorations. Furthermore, it serves as a paradigm of what can be effectively accomplished with the now highly accurate DNA-microarray analysis in generating a robust transcriptional roadmap and in illuminating the physiology of a lesser understood organism.Results and discussionMetabolism and differentiation of C. acetobutylicum: identification of a new cell type?We aimed to relate the metabolic and morphological characteristics of the cells in a typical batch culture, whereby cells underwent a full differentiation program, to the transcriptional profile of the cell population [8]. The metabolism of solventogenic clostridia is characterized by an initial acidogenic phase followed by acid re-assimilation and solvent production [7]. As shown in Figure 1a, the peak of butyrate concentration, around 16 hours after the start of the culture, coincided with the initiation of butanol production. Around this time, the culture transitioned from exponential growth to stationary phase and initiated solventogenesis and sporulation. This period is called the transitional phase and is indicated by the gray bar in Figure 1a and all following figures. The butanol concentration increased to over 150 mM until hour 45, after which no substantial change in solvent or acid concentration took place. Nevertheless, cells continued to display morphological changes well past hour 60. Solventogenic clostridia display a series of morphological forms over this differentiation program: vegetative, clostridial, forespore, endospore, and free-spore forms [9]. In addition to phase-contrast microscopy, we found that by using Syto-9 (a green dye assumed to stain live cells) and propidium iodide (PI; a red dye assumed to stain dead cells) [10] we could microscopically distinguish these morphologies and identify new cell subtypes. Staining by these two dyes did not follow typical expectations. During exponential growth, vegetative cells, characterized by a thin-rod morphology, were visibly motile under the microscope, which is consistent with the finding that chemotaxis and motility genes were highly expressed during this time [7]. When double stained with Syto-9 and PI dyes, these vegetative cells took on a predominantly red color, indicating the uptake of more PI than Syto-9 (Figure 1b, I, II). At the onset of butanol production, swollen, cigar-shaped clostridial-form cells began to appear (Figure 1b, III). These clostridial forms (confirmed by phase-contrast microscopy; data not shown), generally assumed to be the cells that produce solvents [8], were far less motile than exponential-phase cells and stained almost equally with both dyes, taking on an orange color. Clostridial forms persisted until solvent production decreased, after which forespore forms (cells with one end swollen, which is indicative of a spore forming) and endospore forms (cells with the middle swollen, which is indicative of a developing spore) became visible [9]. These cells stained almost exclusively green, indicating an uptake of more Syto-9 than PI (Figure 1b, IV-VI). The sporulation process is completed when the mother cell undergoes autolysis to release the mature spore. Mature free spores could be seen as early as hour 44 (Figure 1b, V). Later, around hour 58 (Figure 1b, VI), a portion of the cells became motile again. Though these cells appear like vegetative cells, they stained predominantly green, instead of red, and did not produce appreciable amounts of acid. We hypothesize that this staining change reflects modifications in membrane composition due to different environmental conditions (presence of solvents and other metabolites) rather than cell viability and assume that this newly identified cell type has different transcriptional characteristics, which we tested next.Figure 1Morphological and gene expression changes C. acetobutylicum undergoes during exponential, transitional, and stationary phases. (a) Growth and acid and solvent production curves as they relate to morphological and transcriptional changes during sporulation. The gray bar indicates the beginning of the transitional phase as determined by solvent production. A600 with microarray sample (filled squares); A600 (open squares); butyrate (filled circles); butanol (filled triangles). Roman numerals correspond with those in (b), and bars and numbers along the top correspond to the clusters in (c). (b) Morphological changes during sporulation. When stained with Syto-9 (green) and PI (red), vegetative cells take on a predominantly red color (I and II). At peak butanol production, swollen, cigar-shaped clostridial-form cells appear (arrow in III), which stain almost equally with both dyes, and persist until late stationary phase. Towards the end of solvent production (IV), endospore (arrow 1) forms are visible, and clostridial (arrow 2) forms are still present. As the culture enters late stationary phase (V and VI), cells stain almost exclusively green, regardless of morphology. All cell types are still present, including free spores (arrows in V and VI), and vegetative cells identified by their motility. (c) Average expression profiles for each K-means cluster generated using a moving average trendline with period 3. (d) Expression of the 814 genes (rows) at 25 timepoints (columns, hours 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 44, 48, 54, 58, and 66). Genes with higher expression than the reference RNA are shown in red and those with lower expression as green. Saturated expression levels: ten-fold difference.The transcriptional program of clostridial differentiationTo ensure that important transcriptional, physiological, and morphological changes were captured [7,8], RNA samples were taken every hour during exponential phase and every two hours after that until late stationary phase when sampling frequency decreased. mRNA from 25 timepoints (Figure 1a) were selected for transcriptional analysis by hybridizing pairs of 22k oligonucleotide microarrays on a dye swap configuration using an mRNA pool as reference. There were 814 genes, or 21% of the genome, that surpassed the threshold of expression in at least 20 of the 25 microarray timepoints and had two or more timepoints differentially expressed at a 95% confidence level [11]; these genes were classified as having a temporal differential expression profile. We chose these strict selection criteria in order to robustly identify the key expression patterns of the differentiation process. We relaxed these criteria in subsequent gene ontology-driven analyses. Expression data were extensively validated by, first, quantitative reverse transcription PCR (Q-RT-PCR) analysis (focusing on key sporulation factors) from a biological replicate culture (Figure 2), and, second, by systematic comparison to our published (but limited in scope and duration) microarray study (see Additional data file 1 for Figure S1 and discussion).Figure 2Q-RT-PCR and microarray data comparison. RNA from a biological replicate bioreactor experiment was reverse transcribed into cDNA for the Q-RT-PCR. All expression ratios are shown relative to the first timepoint for both Q-RT-PCR (open circles) and microarray data (filled squares). Asterisks represent data below the cutoff value for microarray analysis. Samples were taken every six hours starting from hour 6 and continuing until hour 48. The genes examined were from several operons with different patterns of expression.Six distinct clusters of temporal expression patterns were selected (Figure 1c,d) by K-means to achieve a balance between inter- and intra-cluster variability. To examine transcriptional changes in larger functional groups (for example, transcription, motility, translation), each cluster was analyzed according to the Cluster of Orthologous Groups of proteins (COG) classification [12] and the functional genome annotation [13]. To determine if a COG functional group was overrepresented in any of the K-means clusters, first the percentage of each group in the genome was determined, and then the percentage of each group was determined in each of the K-means clusters. By comparing the percentage in the K-means clusters to the genome percentage, we could identify overrepresented groups (Additional data file 2).Exponential phase: motility, chemotaxis, nucleotide and primary metabolismThe first cluster contains 134 genes highly expressed during exponential growth (hours 6 to 10; see Additional data file 2 for a list of the genes). This cluster characterizes highly motile vegetative cells (Figure 1b, I) and, given the minimal amount of knowledge on the genes responsible for motility and chemotaxis in clostridia, our analysis offers the possibility of identifying these genes at the genome scale [14]. This cluster includes the flagella structural components flagellin and flbD, the main chemotaxis response regulator, cheY (CAC0122; responsible for flagellar rotation in B. subtilis \n[15]), as well as several methyl-accepting chemotaxis receptor genes (CAC0432, CAC0443, CAC0542, CAC1600, CAP0048). COG analysis showed that genes related to cell motility (COG class N) and nucleotide transport and metabolism (COG class F) were overrepresented in this cluster (Additional data file 2). In order to investigate cell motility further, all genes that fell within this COG class were hierarchically clustered according to their expression profiles (see Additional data file 3 for Figure S2 and discussion). Interestingly, the two main cell motility gene clusters, the first including most of the flagellar assembly and motor proteins and the second containing most of the known chemotaxis proteins, clustered together and displayed a bimodal expression pattern (Figure S2). The genes were not only expressed during exponential phase but also during late stationary phase, around hour 38, which is consistent with the observation that a motile cell population was again observed in late stationary phase. Included in the category of nucleotide transport and metabolism are several purine and pyrimidine biosynthesis genes: a set of five consecutive genes, purECFMN, the bi-functional purQ/L gene, purA, pyrPR, pyrD, and pyrI. Two other purine synthesis genes (purH, purD) showed very similar profiles but were not classified within this cluster by the clustering algorithm. Vegetative cells, which correspond to this cluster, produce ATP through acidogenesis, whereby the cells uptake glucose and convert it to acetic and butyric acid. Because glucose is the main energy source, multiple genes for glucose transport were included within this cluster, including the glucose-specific phosphotransferase gene, ptsG, the glucose kinase glcK and CAP0131, the gene most similar to B. subtilis glucose permease glcP. The genes required for the metabolism of glucose to pyruvate did not show temporal regulation, suggesting that expression of these genes is constitutive-like (see Additional data file 3 for Figure S3 and discussion). Acetic acid production genes pta and ack were not temporally expressed, but butyrate production genes ptb and buk were. Though expressed throughout exponential phase, the expression of both ptb and buk slightly peaked during late exponential phase, as previously seen [7], and thus fall in the transitional (second) cluster. Analysis of the expression patterns of all the genes involved in acidogenesis, not just the differentially expressed genes discussed here, is included in Figure S3 in Additional data file 3. Finally, the expression patterns of the two classes of hydrogenases (iron only and nickel-iron) were investigated (Figure S3 in Additional data file 3). hydA, the iron only hydrogenase that catalyzes the production of molecular hydrogen, was expressed only during exponential phase, whereas the iron-nickel hydrogenase, mbhS and mbhL, was expressed throughout stationary phase.Initiation of sporulation: abrB, sinR, lipid and iron metabolismThe transitional phase is captured by 139 genes in the second cluster (Figure 1c,d; Additional data file 2). It is made up of genes that show elevated expression between hours 10 and 18 and is when solvent formation was initiated. This cluster characterizes the shift from vegetative cells to cells committing to sporulation and thus includes two important regulators of sporulation, abrB (CAC0310) and sinR (CAC0549), which are discussed in more detail below. Also characteristic of this shift from vegetative growth to sporulation was the overrepresentation of genes related to energy production and conversion (COG class C), since sporulation is an energy intensive process. Solvent production began in the transitional phase, though the genes responsible for solvent production fall in the next (third) cluster; the third cluster partially overlaps with this second cluster but is distinguished by a sustained expression pattern. In response to these solvents, C. acetobutylicum undergoes a change in its membrane composition and fluidity, generally decreasing the ratio between unsaturated to saturated fatty acids [16-18]. Consistent with this change, genes related to lipid metabolism (COG class I) were overrepresented in this cluster. To further investigate this COG class, all genes identified as COG class I were hierarchically clustered (see Additional data file 3 for Figure S4 and discussion). Seven genes that were upregulated just before the onset of sporulation fall within the same operon and are related to fatty acid synthesis. In contrast, many of the most characterized genes involved in fatty acid synthesis (accBC, fabDFZ, and acp) maintain a fairly flat profile throughout the timecourse (Figure S4 in Additional data file 3). Also within this cluster is the gene responsible for cyclopropane fatty acid synthesis (cfa), though classified in COG class M (cell envelope biogenesis) and not COG class I. Importantly, the ratio of cyclopropane fatty acids in the outer membrane has been shown to increase as cells enter stationary phase [18,19], but the overexpression of this gene alone was unable to produce a solvent tolerant strain [19]. Though not overrepresented in this cluster, all the genes within COG class M were also hierarchically clustered (see Additional data file 3 for Figure S5 and discussion). The transitional cluster also included several genes related to iron transport and regulation like the fur family iron uptake regulator CAC2634, the iron permease CAC0788, feoA, feoB, fhuC, and two iron-regulated transporters (CAC3288, CAC3290), which is consistent with the earlier, more limited data [7]. Significantly, iron-limitation has been found to promote solventogenesis [20].Solventogenesis, clostridial form, stress proteins, and early sigma factorsThe third cluster (Figure 1c,d; Additional data file 2) of 175 upregulated genes represents the solventogenic/stationary phase as it contains all key solventogenic genes. This cluster characterizes the transcriptional pattern of clostridial cells, the unique developmental stage in clostridia and first recognizable cell type of the sporulation cascade, and exhibited a longer upregulation of gene expression than the previous two clusters. Indeed, its range overlapped the previous (second) and the next two (fourth and fifth) clusters. The clostridial form is generally recognized to be the form responsible for solvent production [8,21] and is distinguished morphologically as swollen cell forms with phase bright granulose within the cell [21]. This cluster captures both of these characteristics with the inclusion of the solventogenic genes and several granulose formation genes. The solventogenic genes adhE1-ctfA-ctfB, adc, and bdhB were initially induced during transitional phase, the second cluster, but were expressed throughout stationary phase and were thus placed within this cluster. Two granulose formation genes, glgC (CAC2237) and CAC2240, and a granulose degradation gene, glgP (CAC1664), were included within this cluster. The other two granulose formation genes, glgD (CAC2238) and glgA (CAC2239), though not included in this cluster, displayed a similar expression profile to glgC and CAC2240. The concomitant requirement of NADH during butanol production drove the expression of three genes involved in NAD formation: nadABC. Expression of the stress-response gene hsp18, a heat-shock related chaperone, and the ctsR-yacH-yacI-clpC operon, containing the molecular chaperone clpC and the stress-gene repressor ctsR, also fell in this cluster and paralleled the expression of the solventogenic genes (see Additional data file 3 for Figure S6). Other important stress-response genes, groEL-groES (CAC2703-04) and hrcA-grpE-dnaK-dnaJ (CAC1280-83), mirrored this expression pattern, though were not differentially expressed according to the strict criteria employed for selecting the genes of Figure 2c,d (Figure S6 in Additional data file 3). Although genes encoded on the pSOL1 megaplasmid [22] represent less than 5% of the genome, they constitute 15% of genes in this cluster. pSOL1 harbors all essential solvent-formation genes and, importantly, some unknown gene(s) essential for sporulation [22]. Besides the genes listed in this cluster, the vast majority of the genes located on pSOL1 were expressed throughout stationary phase, with most being upregulated at the onset of solventogenesis (see Additional data file 3 for Figure S7). Several key sporulation-specific sigma factors (σF, σE, σG) and the σF-associated anti-sigma factors in the form of the tricistronic spoIIA operon (CAC2308-06) belong to this cluster along with one of the two paralogs of spoVS (CAC1750) and one of three spoVD paralogs (CAP0150). The second spoVS paralog (CAC1817) did not meet the threshold of expression in 12 of the 25 timepoints; the other two paralogs of spoVD (CAC0329, CAC2130) were above the expression cutoff but did not show significant temporal regulation. Of unknown significance was the expression of a large cluster of genes involved in the biosynthesis of the branched-chain amino acids valine, leucine and isoleucine (CAC3169-74) coinciding with the onset of solventogenesis, as shown before [7,23], as well as the upregulation of several glycosyltranferases (see Additional data file 3 for Figure S8). The upregulation of valine, leucine, and isoleucine synthesis genes could be indicative of a membrane fluidity adaptation [7]. In B. subtilis, these branched-chain amino acids can be converted into branched-chain fatty acids and change the membrane fluidity [24], and under cold shock stress, B. subtilis downregulates a number of genes related to valine, leucine, and isoleucine synthesis [25]. Therefore, this upregulation may be another mechanism to change membrane fluidity, though the ratio of unbranched and branched fatty acids has not been reported in studies investigating membrane composition [16-18,26].Stationary phase carbohydrate (beyond glucose) and amino acid metabolismThe fourth cluster (Figure 1c,d; Additional data file 2) of 84 genes represents a sharp induction of expression between 18 and 24 hours (early stationary phase). This cluster falls within the stationary (third) cluster described above. This is a compact group, with 70% belonging to one of three COG categories: carbohydrate transport and metabolism, transport and metabolism of amino acids, and inorganic ion transport and metabolism. A number of different carbohydrate substrate pathways, from monosaccharides (fructose, galactose, mannose, and xylose) to disaccharides (lactose, maltose, and sucrose) to complex carbohydrates (cellulose, glycogen, starch, and xylan), were investigated, and many exhibited upregulation during stationary phase, though only a few are highly expressed (see Additional data file 3 for Figure S9). The significance of this upregulation of non-glucose pathways is unknown, because sufficient glucose remains in the media (approximately 200 mM or about 44% of the initial glucose level). Of particular interest was the upregulation of several genes related to starch and xylan degradation (Figure S9 in Additional data file 3). The two annotated α-amylases (CAP0098 and CAP0168) along with the less characterized glucosidases and glucoamylase were all upregulated throughout stationary phase and a number were highly expressed, like CAC2810 and CAP0098. Also upregulated were the predicted xylanases CAC2383, CAP0054, and CAC1037, with CAP0054 and CAC1037 being highly expressed during stationary phase. Mirroring this pattern were CAC1086, a xylose associated transcriptional regulator, and the highly expressed CAC2612, a xylulose kinase. The genes related to glycogen metabolism are believed to be involved in granulose formation, as discussed earlier. Several genes for arginine biosynthesis (argF, argGH, argDB, argCJ, carB) were induced during this time, probably as a result of its depletion in the culture medium.Genes underlying the activation of the sporulation machinery and the genes for tryptophan and histidine biosynthesisThe fifth cluster (Figure 1c,d; Additional data file 2), representing the middle stationary phase, contains 120 genes mainly expressed between hours 24 and 36, and again falls within the stationary (third) cluster described above. Most of the genes in this cluster activate the sporulation-related sigma factors (σF, σE, σG) or are putatively regulated by them. These include spoIIE, the phosphatase that dephosphorylates SpoIIAA and results in the activation of σF, and the σE-dependent operons spoVR (involved in cortex synthesis), spoIIIAA-AH (required for the activation of σG), and spoIVA (involved in cortex formation and spore coat assembly). The σG-dependent spoVT gene has two paralogs in C. acetobutylicum (CAC3214, CAC3649); the transcriptional pattern suggests that CAC3214, included in this cluster, is the real spoVT. Sporulation-related genes included in this cluster are three cotF genes, one cotJ gene, one cotS gene, the spore maturation protein B, a small acid soluble protein (CAC2365), and two spore lytic enzymes (CAC0686, CAC3244). Though several sporulation-related genes are included in the next (sixth) cluster as well, most, beyond those listed here, are upregulated in mid-stationary phase (see Additional data file 3 for Figure S10 and discussion). Seven genes of the putative operon (CAC3157-63) encoding genes for tryptophan synthesis from chorismate and ten genes for histidine synthesis (CAC0935-43, CAC3031) were also included here.Spore maturation and late-stationary phase vegetative cellsThe sixth cluster, representative of the late stationary phase, includes 162 genes mainly expressed after hour 36 (Figure 1c,d; Additional data file 2). This cluster captured the expression profiles of the forespore and endospore forms, free spores, and late-stage vegetative-like cells. The endospore form represents the last stage before mature spores are released, and therefore fewer sporulation-related genes are within this cluster than previous ones. The sporulation-related genes included in this cluster are two small acid-soluble proteins (CAC1522 and CAC2372), a spore germination protein (CAC3302), a spore coat biosynthesis protein (CAC2190) and a spore protease (CAC1275). Also within this cluster are the two phosphotransferase genes, CAC2958 (a galactitol-specific transporter) and CAC2965 (a lactose-specific transporter), another annotated cheY (CAC2218), various enzymes related to different sugar pathways (CAC2180, CAC2250, CAC2954), and two glycosyltransferases (CAC2172, CAC3049). Expression of these genes may be reflective of the late-stage vegetative-like cells observed during microscopy and demonstrate they have a different genetic profile compared to the early vegetative cells. Interestingly, this cluster is enriched in defense mechanism genes (COG class V) like a phospholipase (CAC3026) and multidrug transporters that may play a role in resistance to a variety of environmental toxins.General processes: cell division and ribosomal proteinsTwo additional gene classes (cell division and ribosomal proteins), though not overrepresented in any of the six clusters described above, were investigated because of their importance in cellular processes and interesting expression patterns. COG class D (cell division and chromosome partitioning), besides important genes for vegetative symmetric division, includes ftsAZ, important for both symmetric and asymmetric cell division, and soj (a regulator of spo0J) and spoIIIE, important for proper chromosomal partitioning between the mother cell and prespore. These genes, along with several uncharacterized genes, were upregulated at the beginning of sporulation (see Additional data file 3 for Figure S11). Almost all the ribosomal proteins were downregulated as the culture entered stationary phase, and interestingly, about half of those downregulated genes were again upregulated in mid-stationary phase and remained upregulated until late-stationary phase (see Additional data file 3 for Figure S12). This upregulation is likely related to the late-stage vegetative-like cells seen.Expression and activity patterns of sporulation-related sigma factors and related genesExpression of sporulation transcription factorsSporulation in bacilli is initiated by a multi-component phosphorelay [27], which is absent in clostridia, but the master regulator of sporulation, Spo0A, is conserved [1,13]. Briefly, in B. subtilis, phosphorylated Spo0A promotes the expression of prespore-specific sigma factor σF and mother cell-specific sigma factor σE [28]. σF is followed by σG, which is controlled by both σF and σE, and σE is followed by σK, which is controlled by σE and SpoIIID [28]. sigH expression, in bacilli, is induced before the onset of sporulation and aids spo0A transcription [28]. Here, sigH expression underwent a modest two-fold induction, relative to the first timepoint, during the onset of sporulation but never increased beyond three-fold, in contrast to all other sporulation factors (Figure 3a). spo0A expression also peaked during the onset of sporulation at over 12-fold and maintained a minimum of 3-fold induction until hour 36 (Figure 3a,b). Once phosphorylated, in bacilli and likely in C. acetobutylicum [29], Spo0A regulates the expression of the operons encoding sigF, sigE, and spoIIE [30], the latter of which acts as an activator of σF. sigF and sigE exhibited an initial 16- and 8-fold induction, respectively, at hour 12, the timing of peak spo0A expression, but a second higher level of induction, 46- and 66-fold, respectively, was reached later at hour 24 (Figure 3c) and confirmed with Q-RT-PCR (Figure 2). The plateau or decrease in expression of spo0A, sigF, and sigE coincided with the peak expression of two known repressors, abrB and sinR, of sporulation genes in B. subtilis (Figure 3b), the former repressing the expression of spo0A promoters and the latter directly binding to the promoter sequences of the spo0A, sigF, and sigE operons [31,32]. C. acetobutylicum contains three paralogs of abrB, among which CAC0310 exhibited the highest promoter activity and, when downregulated, causes delayed sporulation and decreased solvent formation [33]. sinR (CAC0549) expression in C. acetobutylicum was previously reported [33] to be weak, but our data show a significant amount of expression and suggest a similar role as that in B. subtilis. In B. subtilis, Spo0A either indirectly (sinR) or directly (abrB) represses the genes of these two repressors [32,34]. The expression patterns of both genes did decrease after peak Spo0A~P deduced activity (Figure 4b; see below), indicating a similar regulatory network may be involved in C. acetobutylicum. sigF, sigE and sigG have very similar expression patterns (Figure 3c). Both sigF and sigE are activated by Spo0A~P, so similar expression profiles were expected. In B. subtilis, a sigG transcript is also detected early, but this transcript is read-through from sigE, located immediately upstream of sigG, and is not translated [35,36]. Translation of sigG occurs when the gene is expressed as a single cistron from a σF-dependent promoter located between sigE and sigG [35,36]. In C. acetobutylicum, sigE and sigG are also located adjacent to each other, but a σF promoter was not predicted between the two genes [37]. Thus, it was predicted that sigG is only expressed as part of the sigE operon (consisting of spoIIGA, the processing enzyme for σE, and sigE). Our transcriptional data seem to support this prediction because all three genes, spoIIGA, sigE, and sigG, have very similar transcriptional patterns (Figure 3f), suggesting they are expressed as a single transcript, like the spoIIAA-spoIIAB-sigF operon (Figure 3e). However, from Northern blots probing against sigE-sigG, three separate transcripts were seen: one for spoIIGA-sigE-sigG, one for spoIIGA-sigE, and one for sigG [29]. Unfortunately, the current data cannot resolve this issue definitively, since the microarrays only detect if a transcript is present or not.Figure 3Investigation of the sporulation cascade in C. acetobutylicum. (a-f) Expression profiles of sporulation genes shown as ratios against the first expressed timepoint. (a) The first three sporulation factors: spo0A (red filled triangles), sigH (black filled squares), and sigF (open blue circles). (b) spo0A (red filled triangles) and possible sporulation regulators: abrB (open black circles) and sinR (green filled diamonds). (c) Sporulation factors downstream of spo0A: sigF (open blue circles), sigE (black filled triangles), and sigG (open red squares). (d) Genes related to sigK expression: spoIIID (blue filled diamonds), yabG (red filled triangles), and spsF (black filled triangles). (e) spoIIA operon: spoIIAA (black filled diamonds), spoIIAB (red filled triangles), and sigF (open blue circles). (f) spoIIG operon and sigG: spoIIGA (green filled diamonds), sigE (black filled triangles), and sigG (open red squares). The gray bar indicates the onset of transitional phase. (g) Ranked expression intensities. White denotes a rank of 1, while dark blue denotes a rank of 100 (see scale). Gray squares indicate timepoints at which the intensity did not exceed the threshold value. Bracketed genes are predicted to be coexpressed as an operon.Deduced activity profiles of sporulation factorsWe also desired to estimate the activity profiles for the key sporulation factors (σH, Spo0A, σF, σE, and σG; Figure 4). We did so by averaging the expression profiles of known or robustly identifiable canonical genes of their regulons [1]. To adjust for differences in relative expression levels, expression profiles were standardized before averaging [7]. This is a surrogate reporter assay, which we believe is as accurate as most reporter assays. For a detailed discussion of the genes used to construct the plots, see Additional data file 4. For all of the plots (Figure 4), peak activity took place after peak expression, as expected. Of all the factors, σH activity peaked first, during early transitional phase, and this was followed by a decrease in activity until stationary phase, when activity increased again (Figure 4a,f). Spo0A~P activity was the next to peak, during late transitional phase, and stayed fairly constant throughout the rest of the timecourse (Figure 4b,f). σF activity had an initial induction during transitional phase, but then stayed constant until 24 hours (Figure 4c,f). After 24 hours, the activity increased again and stayed fairly constant at this higher activity level for the rest of the culture. σE activity increased slightly during late transitional phase, but its major increase occurred after 24 hours during mid-stationary phase (Figure 4d,f). Like the previous sigma factors, σG activity increased throughout early stationary phase and early mid-stationary phase, but the major increase occurred after hour 30 (Figure 4e,f). The activity of all of the factors, except for Spo0A and σF, decreased during late stationary phase at hour 38. σG activity began to increase slightly again at hour 48 but did not peak again. Considering only major peaks in activity, the Bacillus model of sporulation is generally true with the peaks progressing from σH to Spo0A~P to σF to σE and finally to σG (Figure 4f).Figure 4Transcriptional and putative activity profiles for the major sporulation factors. The standardized expression ratios compared to the RNA reference pool of (a) sigH, \n(b) spo0A, \n(c) sigF, \n(d) sigE, and \n(e) sigG are shown in black, while the activity profiles based on the averaged standardized profiles of canonical genes under their control are shown in red. Putative genes (based on the B. subtilis model) responsible for activating σF (spoIIE), σE (spoIIR), and σG (spoIIIA operon) are shown as light blue diamonds. For the spoIIIA operon, the individual standardized ratios (Figure S13g in Additional data file 4) were averaged together. The gray bar indicates the onset of the transitional phase. (f) Compilation of the activity profiles for sigH (red), spo0A (blue), sigF (green), sigE (black), and sigG (purple). The numbers along the top correspond to the clusters in Figure 1c,d and the bars indicate the timing of each cluster.Can we deduce the activation and processing of σF, σE, and σG from transcriptional data?In B. subtilis, the sigma factors downstream of Spo0A (σF, σE, and σG) are all regulated by a complex network of interactions [1]. We desired to examine if our transcriptional data could be used to do a first test to determine whether the mechanisms employed in the B. subtilis model are valid for C. acetobutylicum. In B. subtilis, σF is held inactive in the pre-divisional cell by the anti-σF factor SpoIIAB. σF is released when the anti-anti-σF factor SpoIIAA is dephosphorylated by SpoIIE, resulting in SpoIIAA binding to SpoIIAB, which then releases σF. In C. acetobutylicum, spoIIAB (CAC2307) and spoIIAA (CAC2308) are transcribed on the same operon as sigF (Figure 3e), but spoIIE (CAC3205) is transcribed separately. The initial increase in σF activity during the transitional phase was not accompanied by an increase in spoIIE expression, but the peak in σF activity did occur after spoIIE upregulation (Figure 4c). Despite the sustained level of σF activity, sigF and spoIIE decreased in expression, though spoIIE expression did increase slightly again after 48 hours (Figure 4c). In B. subtilis, the pro-σE translated from the sigE gene undergoes processing from SpoIIGA, which must interact with SpoIIR in order to accomplish the σE activation. In C. acetobutylicum, SpoIIGA (CAC1694) is transcribed on the same operon as sigE (Figure 3f), and SpoIIR is coded by CAC2898. σE activity increased with the induction of spoIIR (Figure 4d), suggesting a similar mechanism as in B. subtilis. Finally, σG activation in B. subtilis is dependent upon the eight genes within the spoIIIA operon. Here, the second and larger increase in σG activity followed peak expression of the spoIIIA operon, but the early increase in σG activity was not characterized by a large induction of spoIIIA expression (Figure 4e). We tentatively conclude that the B. subtilis processing and activation model does generally hold true in C. acetobutylicum, but further investigation is needed to determine the exact timing and interaction of the various factors and their activators.Is there a functional sigK?In B. subtilis, σK is formed by splicing together two genes (spoIVCB and spoIIIC), both under the control of σE and SpoIIID [38], separated by a skin element [39]. In contrast, a single gene encoding σK has been annotated in C. acetobutylicum [13]. The gene was initially identified using a PCR-approach [40] and was later detected by primer extension in a phosphate-limited, continuous culture of C. acetobutylicum DSM 1731 [41]. spoIIID, which controls sigK expression with σE in B. subtilis, reached peak expression at hour 30, which is consistent with it being under σE control (Figure 3d) [42]. However, at no timepoint in this study did sigK exceed the cutoff expression criterion. Q-RT-PCR also showed a significantly lower sigK induction compared to the other sigma factors and suggests the transcript, if expressed, is at much lower levels than any other gene analyzed (Figure 2). The putative main σK processing enzyme, SpoIVFB (CAC1253), also did not exceed the cutoff criterion. To help determine if there is an active σK, we investigated two genes controlled by σK in B. subtilis. yabG (CAC2905), which encodes a protein involved in spore coat assembly, was upregulated mid-stationary phase and peaked at hour 30 (Figure 3d), and spsF (CAC2190), involved in spore coat synthesis, was not upregulated until late stationary phase, at hour 38 (Figure 3d). From these two genes, it is difficult to determine whether a functional sigK gene exists or not. Clearly they are both transcribed, but based on its expression pattern, yabG could fall under the control of σE instead of σK. spsF upregulation is late enough to possibly indicate σK regulation though. Ideally, more genes need to be investigated to draw firmer conclusions, but because few σK regulon homologs exist in C. acetobutylicum, we cannot currently determine if there is σK activity or not.Distinct profiles of sensory histidine kinases: which for Spo0A?Revisiting the orphan kinasesAs discussed, phosphorylated Spo0A is responsible for initiating sporulation in both bacilli and clostridia along with solvent formation in C. acetobutylicum. In bacilli, Spo0A is phosphorylated via a multi-component phosphorelay [43], initiated by five orphan histidine kinases, KinA-E (kinases that lack an adjacent response regulator); this phosphorelay system is absent in all sequenced clostridia [1]. Alternatively, Spo0A in clostridia may be directly phosphorylated by a histidine kinase, orphan or not, as was hypothesized in [1,7]. This alternative was demonstrated in C. botulinum, where the orphan kinase CBO1120 was able to phosphorylate Spo0A [44]. In C. acetobutylicum, five true orphan kinases have been identified with a sixth orphan, CAC2220, identified as CheA, which has a known response regulator [1].A kinase that could directly phosphorylate Spo0A is expected to have a peak in expression before or during the activation of Spo0A, as the orphan kinases in B. subtilis do [45-47]. As a measure of Spo0A activity, the expression of the sol operon (CAP0162-64) was used, as before [7], because it is induced by Spo0A~P. The initial induction of the sol operon, almost 100-fold, occured at hour 10 (before spo0A reached it maximum expression), with detectable levels of butanol appearing before the second induction of the sol operon. This second induction, of another 10-fold, followed the peak in spo0A expression (Figure 5a). It is clear that some level of phosphorylated Spo0A exists at 10 hours; therefore, kinase candidates must display an increase in expression before 10 hours. Of the five orphan kinases (Figure 5b,c), CAC2730 displayed the earliest peak followed by CAC0437, CAC0903, and CAC3319. CAC0323 never displayed a prominent peak in expression either before or after sol operon induction (Figure 5b) and likely does not play a role in phosphorylating Spo0A. Of the remaining four, CAC0437 and CAC2730 peaked only once before the initial sol operon induction, while CAC0903 peaked before each induction of the sol operon (Figure 5b,c). CAC3319 expression slightly mirrored that of the sol operon, with an increase before initial induction followed by a plateau, and an increase in expression again until it peaked just after the sol operon peaked (Figure 5c). The proteins encoded by CAC0437 and CA0903 displayed the most similarity to the protein encoded by CBO1120, the orphan kinase in C. botulinum shown to phosphorylate Spo0A [44].Figure 5Expression profiles of uncharacterized sensory histidine kinases that could phosphorylate Spo0A. Gene and operon profiles are ratios compared against the first expressed timepoint. Gray bar indicates the onset of the transitional phase. (a) Activation of Spo0A as represented through the upregulation of the sol operon (black filled diamonds; CAP0162-164) and the production of butanol (green crosses). Activation occurs before spo0A (red filled triangles) reaches peak expression. (b) Expression of the orphan kinases CAC0323 (blue filled diamonds), CAC0437 (green filled triangles), and CAC0903 (red filled circles) relative to the sol operon (black filled diamonds) (right-hand side vertical axis). (c) Expression of the orphan kinases CAC2730 (blue filled squares) and CAC3319 (open red circles) relative to the sol operon (black filled diamonds) (right-hand side vertical axis). (d) Expression of the two-component kinases CAC0225 (green filled circles), CAC0290 (red filled squares), and CAC0863 (open blue diamonds) relative to the sol operon (black filled diamonds) (right-hand side vertical axis). (e) Expression of the two-component kinases CAC1582 (green filled squares), CAC2434 (open blue circles), and CAC3430 (open red diamonds) relative to the sol operon (black filled diamonds) (right-hand side vertical axis). (f) Ranked expression intensities. White denotes a rank of 1, while dark blue denotes a rank of 100 (see scale). Plot covers the entire timecourse, whereas the previous figures only covered the first 14 hours. Gray squares indicate timepoints at which the intensity did not exceed the threshold value.Non-orphan kinase expressionThough primarily interested in orphan kinases because of the similarity to the B. subtilis model, a two-component response system could also be responsible for the phosphorylation of Spo0A. The remaining 30 annotated histidine kinases were also investigated to determine if any displayed a peak in expression before the initial induction of the sol operon (Additional data file 5). Six kinases (Figure 5d,e) were found to have a peak in expression at 8 hours. CAC0290 and CAC3430 subsequently decreased in expression while CAC0225 and CAC0863 maintained expression at initial levels. Despite a dip in expression at hour 9, CAC1582 maintained an increased expression level from 8 hours on. CAC2434 peaked at hour 8, dropped back to initial levels, but then steadily increased with the second induction of the sol operon.Sigma factors of unknown function: a first assessment of their functional rolesSeventeen sigma factors are annotated on the C. acetobutylicum genome, including two on pSOL1. Two, sigK (CAC1689) and CAC1770 (a sigK-like sigma factor), are expressed at very low levels and two others, CAC1509 (annotated 'specialized sigma subunit of RNA polymerase') and CAC1226 (one of two annotated sigAs), are only above the expression cutoff in 8 out of 25 timepoints, and these timepoints are not consecutively expressed. Among the expressed sigma factors, six, CAP0157, CAP0167, CAC3267, CAC1766, CAC2052, and CAC0550, are of unknown function, while the remaining seven expressed sigma factors (σH, σF, σE, σG, σA, σD, and σ54/rpoN) are of predicted known function. To assess the potential role of the remaining six sigma factors of unknown function, we examined the transcriptional profiles (Figure 6a,b) and probed the binding motifs in their promoter regions for predicted Spo0A, σA, σE, and σF/σG binding motifs [37].Figure 6Expression profiles of sigma factors with unknown function and the effects of down-regulation. (a) Expression profiles of CAC3267 (open triangles), CAP0167 (filled squares), and CAP0157 (open circles) as ratios compared to the first expressed timepoint. Gray bar indicates the onset of transitional phase. (b) Expression profiles of CAC0550 (filled circles), CAC2052 (open squares), and CAC1766 (filled triangles) as ratios compared to the first expressed timepoint. Gray bar indicates the onset of transitional phase. (c) Ranked expression intensities of the sigma factors. White denotes a rank of 1, while dark blue denotes a rank of 100 (see scale). Gray squares indicate timepoints at which the intensity did not exceed the threshold value. (d) Microscopy time-course of asRNA strains compared to WT and plasmid control strains. Microscopy samples from WT (I) and pSOS95del (II) cultures (as controls) and three asRNA strains taken for two timepoints over a course of 72 hours. At 72 hours, WT (I) and pSOS95del (II) exhibit the typical clostridial forms (white arrows), while asCAP0166 (III) shows advanced differentiation with forespores and endospores (orange arrows) already visible. Strains asCAP0166 (III), asCAP0167 (IV), and asCAC1766 (V) show a novel, extra-swollen clostridial form (yellow arrows).Transcriptional analysis of the sigma factors of unknown functionLoss of pSOL1 impairs sporulation at the level of spo0A expression [7,48], thus generating increased interest for sigma factors located on the pSOL1 plasmid as these may play a role in the regulation of sporulation. Two sigma factors, CAP0157 and CAP0167, are located on pSOL1 and are annotated as 'special sigma factor (σF/σE/σG family)' and 'specialized sigma factor (σF/σE family)', respectively. It was predicted that CAP0167 is putatively co-transcribed with CAP0166 from a promoter of the σF/σG family [37] and it displayed an expression pattern similar to that of spo0A, consistent with the computational prediction of an 0A box [29] and two reverse 0A boxes in its promoter region (Figure 6a). CAP0157 was expressed from an unidentified promoter late in the timecourse (40+ hours) and thus may be involved in late-stage sporulation, despite its low level of expression at hour 20 (Figure 6a). CAC3267, putatively the fourth gene in an operon starting with CAC3270 and ending with CAC3264 [37], was mainly expressed during early exponential growth (Figure 6a), then decreased, and peaked again around 14 hours, after which expression decreased again. This pattern of expression suggests that it plays a role in vegetative growth and possibly early sporulation. CAC0550, putatively transcribed from a σA promoter as a single cistron [37], was mainly transcribed early with its expression ending after 20-24 hours (Figure 6b), suggesting that it is not involved in sporulation. CAC1766, expressed from an unknown promoter, displayed a unique pattern with a progressive buildup starting around hours 8-12 and a distinct peak around hour 22 (Figure 6b). CAC2052 is annotated as 'DNA-dependent RNA polymerase σ-subunit' and was putatively expressed together with CAC2053, a hypothetical protein, from a σA and/or a σF/σG promoter [37]. Our data suggest that it is unlikely to be transcribed from a σF/σG promoter without any other effectors, as their transcription peaked at hour 16, when there was very little (if any) σF or σG activity (Figure 6b).Phylogenetic tree comparisonTo help determine a possible function for these sigma factors, a phylogenetic tree was constructed of σ70 sigma factors from ten species, including B. subtilis and all sequenced clostridial species. The resulting tree (Additional data file 6) contains eleven major branches, and of these, seven can be definitively classified based on known sigma factors within the branch. These categories are extracytoplasmic function (ECF), sporulation factors (sigF, sigE, and sigG), sigH, sigA (a basal sigma factor), sigD (regulates chemotaxis and motility), and sigB (a general response sigma factor). Two factors, CAC3267 and CAC1766, fell within ECF branches. CAC3267 fell within an ECF branch close to the B. subtilis σV, a sigma factor of unknown function, and σM, a sigma factor essential for growth and survival in high salt concentrations. CAC1766 fell within a different ECF branch close to B. subtilis σZ, a sigma factor of unknown function, and CAC1509, a sigma factor expressed for less than eight consecutive timepoints. The remaining four factors fell within clusters with other clostridial sigma factors of unknown function, though several could have possible ECF function.Antisense RNA knock-down of four sigma factors: 'fat' clostridial forms and enhanced glucose metabolismOf the six expressed sigma factors of unknown function, CAP0157, CAP0167, CAC2052, and CAC1766 were chosen for further study because the timing and shape of their expression patterns suggested potential involvement in sporulation and/or solventogenesis. Since the two processes are coupled, phenotypic changes in differentiation may affect solvent production, as has been previously observed [4,6,29,33,49]. Antisense RNA (asRNA) knock-down was chosen over knocking out the genes, because knockouts are still extremely difficult to produce in this and all other clostridia. Indeed, to date, only a handful of knockouts have been created [29,50-53], and these have only been achieved after screening thousands of transformants [51-53]. Recently, a group II intron system has been developed for clostridia [54], but this system was not yet available when these experiments were carried out. In contrast, asRNA is relatively quick, has been shown to reduce gene expression by up to 90% [33,55,56] and has been used to knock-down a large number of genes with a high level of specificity [33,49,55-59]. asRNA constructs (see Additional data file 7 for specific sequences used) were designed against CAP0157, CAP0167, CAC2052, and CAC1766 along with CAC2053 and CAP0166, the first genes in the operons predicted to contain CAC2052 and CAP0167, respectively [37]. Cultures of these strains were examined and compared against the wild type (WT) and plasmid control strain 824(pSOS95del) for cell morphology differences and metabolic changes.Microscopy results from the asRNA-strain cultures revealed both novel morphologies and apparently altered differentiation (Figure 6d). Most notable were changes in strains asCAP0166, asCAP0167 and asCAC1766. Typical WT cultures display a predominately vegetative, symmetrically dividing population through 72 hours as evidenced by the thin, rod-shaped, phase dark cells (Figure 6d, I). By 72 hours, WT cultures exhibited only a small percentage of swollen, cigar-shaped clostridial forms and then a proportional population of free spores by 96 hours.pSOS95del cultures exhibited clostridial forms by 48 hours, suggesting an accelerated differentiation compared to WT, as has been seen before in our laboratory (Figure 6d, II). Moreover, a greater percentage of clostridial forms and free spores compared to WT were observed at 72 and 96 hours, respectively. asCAP0166 cultures generated a large percentage of clostridial forms and endospores/free spores by hours 48 and 72, respectively (Figure 6d, III). This differentiation is accelerated in comparison to pSOS95del. By hour 96, asCAP0166 cultures exhibited predominately vegetative cells apparently derived from germinated spores (data not shown). asCAP0167 cultures also exhibited accelerated differentiation and displayed a novel (to our knowledge) form of cellular morphology that was most profoundly observable at 72 hours (Figure 6d, IV). This novel morphology has qualities of an excessively swollen clostridial cigar-form (which makes them look much shorter than normal clostridial forms), with what appears to be endospore formation occurring, but without the associated phase bright characteristics seen in the 72 hour asCAP0166 cultures. The asCAP0166 culture displayed cells in this novel morphological state as well, but to a lesser extent, although it is possible that because of its faster sporulation, such cell forms appeared prior to 72 hours. The asCAC1766 cultures also exhibited altered differentiation; most importantly, at 72 hours the majority of the cells exhibited a very swollen clostridial-form morphology similar to that in the asCAP0167 cultures at 72 hours, but slightly more elongated (Figure 6d, V).To further characterize this novel cell form, transmission electron microscopy (TEM) and scanning electron microscopy images of cells were taken for strains asCAP0167 and asCAC1766. To determine morphological differences involved in differentiation, the TEM images were compared against cell images taken from the plasmid control strain (Figure 7). For both asRNA strains, the very swollen cell forms observed can be documented as approximately 2.5-4 μm long, and 1.1-1.3 μm in diameter, and should be compared to control or WT swollen clostridial forms, which are 3.5-6 μm long and 0.8-1 μm in diameter. Forespore and endospore forms of both asCAP0167 (Figure 7c,d) and asCAC1766 (Figure 7e,f) displayed a pinched end not seen in the plasmid control (Figure 7b). A slight pinching is seen in the clostridial forms of the plasmid control strain (Figure 7a), but this is probably indicative that an asymmetric division is about to occur. Rather, the pinched ends seen in the antisense strains occur after asymmetric division and while the spore is developing within the mother cell. These pinched ends are also noticeable in the scanning electron microscopy images (Figure 8). Though granulose is distinguishable in most of the TEM images (Figure 7c,d,f), it is not the characteristic electron translucent seen in typical clostridial, forespore, and endospore forms (Figure 7a,b). These differences were seen throughout the culture and additional TEM images of both the plasmid control and the antisense strains are included in Additional data file 8.Figure 7TEM images of the novel cell forms. (a-b) TEM images of the plasmid control strain pSOS95del: typical elongated clostridial form with electron translucent granulose (a); typical endospore form with a developing endospore at one end of the cell and electron translucent granulose still visible at the other end of the cell (b). (c-d) TEM images of the antisense strain asCAP0167. (e-f) TEM images of the antisense strain asCAC1766. Red arrows in (c-f) indicate pinched portions of the cell membrane not seen in the control strain and are characteristic of this novel cell type. Also noticeable is the electron dense granulose in the antisense strains, in contrast to the electron translucent granulose in the control samples.Glucose, acetone, and butanol concentrations from two to four biological replicates for each strain were averaged together, and the results are shown in Table 1. We averaged data from cultures that displayed similar characteristics; most cultures did so despite the fact that each culture was inoculated from a different colony for each strain. Acetone and butanol levels were typical for WT and control cultures, with the WT producing 90 mM of acetone and 150 mM of butanol and the plasmid-control strain producing 80 mM of acetone and 160 mM of butanol [60]. By 192 hours, all strains had either produced comparable amounts of butanol to the WT and the plasmid control strain or had somewhat outperformed these two strains. The most significant differences were that all asRNA strains consumed higher levels of glucose and also had a delayed metabolism in terms of product formation. These metabolic changes, although preliminary, are consistent with and support the large changes in the kinetics of sporulation observed by microscopy.Table 1Concentrations of glucose, acetone, and butanol for asRNA strains96 hours144 hours192 hours*SampleGlucose†Acetone†Butanol†Glucose†Acetone†Butanol†Glucose†Acetone†Butanol†Wild type165911571437415712061162pSOS95del‡26457971368316912557158asCAC1766274678411812316911497163asCAC205229449691918412211692154asCAC20532855477158941429488161asCAP015731449631989112296111174asCAP016629055771181251677791176asCAP01672945473781251805698185*At 192 hours, significant amounts of acetone had evaporated along with small amounts of butanol. However, the cultures were still metabolically active, as indicated by the decreased amounts of glucose and increased amounts of butanol. †Concentrations are mM. ‡pSOS95del was used as a plasmid control strain.ConclusionThis detailed and previously unrevealed transcriptional roadmap has allowed for the first time a complete investigation of the genetic events associated with clostridial differentiation. We were able to link distinct and striking global transcriptional changes to previously known important morphological and physiological changes. To date, this is the most complete genetic analysis of the different morphological forms: vegetative, clostridial, and forespore/endospore. Importantly, this analysis was performed on a mixed culture, which may either dilute or produce noise in the data, but investigation of the clusters identified revealed that these clusters do capture important known processes. We were also able to identify a cell population late in the timecourse similar to vegetative cells. Visually, these late cells looked and acted like vegetative cells, and transcriptionally, they were also fairly similar. The major cell motility and chemotaxis genes were upregulated both early and late in the timecourse (Figure S2 in Additional data file 3), as were the ribosomal proteins (Figure S12 in Additional data file 3). Also, the cell division associated genes rodA, ftsE, and ftsX follow the same transcriptional pattern of both early and late expression (Figure S11 in Additional data file 3). Although, these cells stain differently from the early vegetative cells, probably due to changes in membrane structure in response to the presence of solvents and do not produce detectable levels of acids or solvents, we believe these cells are germinated cells from spores produced early in the timecourse. While the triggers for both sporulation and germination are not known [1], the culture late in the timecourse is less acidic because of the acid reassimilation, and pH has been shown to be a trigger for sporulation [21].This study has also allowed the first full comparison to the widely studied B. subtilis sporulation program. We have confidently identified the temporal orchestration of all known sporulation-related transcription factors and conclude the Bacillus model generally holds true with the cascade progressing in the following manner: σH, Spo0A, σF, σE, and σG (Figure 4f). In addition, we can conclude that the major activating/processing proteins involved in sigma factor activation in B. subtilis play a similar role in C. acetobutylicum, though additional investigation is needed to clarify their role. Of significance is the lack of sigK signal. The genes responsible for transcribing sigK in B. subtilis, sigE and spoIIID, were expressed, but the putative processing enzyme spoIVFB was not. Two genes under the control of σK in B. subtilis were expressed, but their expression patterns are not consistent with each other. Based on the expression pattern of yabG, it could be controlled by σE, while the late expression of spsF could be an indication of σK activity.Finally, in order to determine if one of the annotated sigma factors of unknown function could be a sigK-like gene, we first investigated their transcriptional profiles. CAP0157 was a possible candidate with its upregulation late in the timecourse, as was CAC1766 since its expression was sustained throughout the stationary phase (Figure 6a,b). Neither of these genes, nor any of the other sigma factors of unknown function, clustered close to the known sporulation-related sigma factors on the phylogenetic tree (Additional data file 6), but when downregulated using asRNA, both CAC1766 and the CAP0167 operon (CAP0166 and CAP0167) displayed altered differentiation (Figures 6d, 7 and 8). Though involved in differentiation, the exact role of these two sigma factors is difficult to assess because of the incomplete silencing of the genes through asRNA downregulation. Mature free spores and typical endospore forms without a pinched end are still seen (data not shown), but whether these develop from the novel cell types or from cells not affected by the antisense cannot be determined. Interestingly, both CAP0167 and CAC1766 clustered together with other clostridial sigma factors and closer to ECF sigma factors than to the major sporulation sigma factors sigF, sigE, and sigG (Additional data file 6). In B. subtilis, ECF sigma factors do not play a role in differentiation [61,62], though a triple mutant in sigM, sigW, and sigX did display altered phenotypes [62]. The fact that CAC1766 and CAP0167 appear to affect the developmental process of sporulation (Figures 7 and 8; Additional data file 8) suggests either that ECF factors may play a role in sporulation in clostridia or that a novel category of sigma factors exist in clostridia that play a role in sporulation.Figure 8Scanning electron microscopy (SEM) images of the novel cell forms. SEM images of the antisense strains (a-c) asCAP0167 and (d-f) asCAC1766. Red arrows in indicate pinched portions of the cell membrane not seen in the control strain and are characteristic of this novel cell type.Materials and methodsFermentation analysisTwo cultures of C. acetobutylicum ATCC 824 were grown in pH controlled (pH >5) bioreactors (Bioflow II and 110, New Brunswick Scientific, Edison, NJ, USA) [7]. Cell density, substrate and product concentrations were analyzed as described [56].RNA isolation and cDNA labelingSamples were collected by centrifuging 3-10 ml of culture at 5,000×g for 10 minutes, 4°C and storing the cell pellets at -85°C. Prior to RNA isolation, cells were washed in 1 ml SET buffer (25% sucrose, 50 mM EDTA [pH 8.0], and 50 mM Tris-HCl [pH 8.0]) and centrifuged at 5,000×g for 10 minutes, 4°C. Pellets were processed similarly to [7] but with the noted modifications. Cells were lysed by resuspending in 220 μl SET buffer with 20 mg/ml lysozyme (Sigma, St. Louis, MO, USA) and 4.55 U/ml proteinase K (Roche, Indianapolis, IN, USA) and incubated at room temperature for 6 minutes. Following incubation, 40 mg of acid-washed glass beads (≤106 μm; Sigma) were added to the solution, and the mixture was continuously vortexed for 4 minutes at room temperature. Immediately afterwards, 1 ml of ice cold TRIzol (Invitrogen, Carlsbad, CA, USA) was added; 500 μl of sample was diluted with an equal volume of ice cold TRIzol and purified. Following dilution, 200 μl of ice cold chloroform was added to each sample, mixed vigorously for 15 s, and incubated at room temperature for 3 minutes. Samples were then centrifuged at 12,000 rpm in a tabletop microcentrifuge for 15 minutes at 4°C. The upper phase was saved and diluted by adding 500 μl of 70% ethanol. Samples were then applied to the RNeasy Mini Kit (Qiagen, Valencia, CA, USA), following the manufacturer's instructions. To minimize genomic DNA contamination, samples were incubated with the RW1 buffer at room temperature for 4 minutes. The method disrupted all cell types equally, as evidenced by microscopy (data not shown). cDNA was generated and labeled as described [7]. The reference RNA pool contained 25 μg of RNA from samples taken from the same culture at 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 44, 48, 54, 58, and 66 h.Microarray analysisAgilent technology 22k arrays, (GEO accession number GPL4412) as described in [63], were hybridized, washed, and scanned per Agilent's recommendations. Spot quantification employed Agilent's eXtended Dynamic Range technique with gains of 100% and 10% (Agilent's Feature Extraction software (v. 9.1)). Normalization and slide averaging was carried out as described [7,63]. A minimum intensity of 50 intensity units was used as described [63]. Microarray data have been deposited in the Gene Expression Omnibus database under accession number GSE6094. To gain a qualitative measure of the abundance of an mRNA transcript, the averaged normalized log mean intensity values were ranked on a scale of 1 (lowest intensity value) to 100 (highest intensity value). Genes were clustered using TIGR's MEV program [64].Quantitative RT-PCRQ-RT-PCR was performed as described [48]. Specific primer sequences are included in Additional data file 9; CAC3571 was used as the housekeeping gene.MicroscopyFor light microscopy, samples were stored at -85°C after 15% glycerol was added to the sampled culture. Samples were then pelleted, washed twice with 1% w/v NaCl and fixed using 50 μl of 0.05% HCl/0.5% NaCl solution to a final count of 106 cells/μl. Slides were imaged using a Leica widefield microscope with either phase contrast or Syto-9 and PI dyes (Invitrogen LIVE/DEAD BacLight Kit) to distinguish cell morphology.For electron microscopy, samples were fixed by addition of 16% paraformaldehyde and 8% glutaraldehyde to the culture medium for a final concentration of 2% paraformaldehyde and 2% glutaraldehyde. For cultures grown on plates, colonies were scraped from the agar and suspended in 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4). Cultures were fixed for 1 h at room temperature, pelleted and resuspended in buffer.For transmission electron microscopy, bacteria were pelleted, embedded in 4% agar and cut into 1 mm × 1 mm cubes. The samples were washed three times for 15 minutes in 0.1 M sodium cacodylate buffer (pH 7.4), fixed in 1% osmium tetroxide in buffer for 2 h, and then washed extensively with buffer and double de-ionized water. Following dehydration in an ascending series of ethanol (25, 50, 75, 95, 100, 100%; 15 minutes each), the samples were infiltrated with Embed-812 resin in 100% ethanol (1:3, 1:2, 1:1, 2:1, 3:1; 1 h each) and then several changes in 100% resin. After an overnight infiltration in 100% resin, the samples were embedded in BEEM capsules and polymerized at 65°C for 48 h. Blocks were sectioned on a Reichert-Jung UltracutE ultramicrotome and ultrathin sections were collected onto formvar-carbon coated copper grids. Sections were stained with methanolic uranyl acetate and Reynolds' lead citrate [65] and viewed on a Zeiss CEM 902 transmission electron microscope at 80 kV. Images were recorded with an Olympus Soft Imaging System GmbH Megaview II digital camera. Brightness levels were adjusted in the images so that the background between images appeared similar.For scanning electron microscopy, fixed samples were incubated on poly-L-lysine coated silica wafers for 1 h and then rinsed three times for 15 minutes in 0.1 M sodium cacodylate buffer (pH 7.4). The samples were fixed with 1% osmium tetroxide in buffer for 2 h, washed in buffer and double de-ionized water, and then dehydrated in ethanol (25, 50, 75, 95, 100, 100%; 15 minutes each). The wafers were critical point dried in an Autosamdri 815B critical point drier and mounted onto aluminum stubs with silver paint. The samples were coated with Au/Pd with a Denton Bench Top Turbo III sputter-coater and viewed with a Hitachi 4700 FESEM at 3.0 kV.Phylogenetic tree generationBased on the genome annotations available at NCBI, we considered any sigma factor that was annotated as σ70 or unannotated. A second filter was applied by requiring that all the sequences should contain a Region 2, the most conserved region of the σ70 protein. All members of this class of sigma factor contain Region 2, and it was modeled with the HMM pfam04542. This criterion removed CAC0550, CAC1766 and CAP0157, but they were added to the list again despite their lack of a Region 2. The alignment was made using ClustalW 1.83 using the default settings and visualized as a radial tree as created by Phylodraw v. 0.8 from Pusan National University.Generation and characterization of antisense strainsOligonucleotides were designed to produce asRNA complementary to the upstream 20 bp and first 30-40 bp of the targeted genes' transcripts (Additional data file 7). The constructs were cloned into pSOS95del under the control of a thiolase (thl) promoter and confirmed by restriction digest. Plasmids were then methylated and transformed into C. acetobutylicum ATCC 824, as previously described [33,55,56]. Strains were grown in 10 ml cultures and characterized using microscopy and HPLC to analyze final product concentrations [56].AbbreviationsasRNA, antisense RNA; COG, Cluster of Orthologous Groups; ECF, extracytoplasmic function; PI, propidium iodide; Q-RT-PCR, quantitative reverse transcription PCR; TEM, transmission electron microscopy; WT, wild type.Authors' contributionsSWJ carried out the microarray experiments, helped with the electron microscopy, helped analyze the data, and drafted and finalized the manuscript. CJP designed the microarray platform used, helped with the bioinformatic tools used in the analysis, and drafted parts of the manuscript. BT carried out all the microscopy except the electron microscopy and generated the antisense RNA strains. NC carried out the microarray experiments and helped with the generation of the antisense strains. RS helped design the microarray experiments, carried out the Q-RT-PCR experiments, helped analyze the data, and drafted parts of the manuscript. RSS helped with the bioinformatic tools used in the analysis. ETP helped in the design of all the experiments, the analysis and interpretation of the data, and helped in the organization, draft and editing of the manuscript. All authors read and approved the final manuscript.Additional data filesThe following additional data are available. Additional data file 1 is a figure comparing the present microarray study to an earlier microarray study that examined the early sporulation of C. acetobutylicum followed by a brief discussion. Additional data file 2 contains tables detailing the COG analysis for each cluster and all the genes placed in each cluster. Additional data file 3 contains figures of the transcriptional profiles, in terms of both intensity and differential expression, of specific gene clusters with brief discussions following several figures. Additional data file 4 is a composite figure showing the individual expression profiles of the genes that were standardized and averaged and is followed by a brief discussion on how the genes used to construct the deduced activity plots were chosen. Additional data file 5 is a figure showing the differential expression and intensity of all annotated histidine kinases and response regulators. Additional data file 6 is a figure showing the phylogenetic tree resulting from the alignment of the σ70-related and unannotated sigma factors from ten bacterial species. Additional data file 7 is a table listing the sequences for each asRNA construct. Additional data file 8 contains figures showing additional TEM images of the plasmid control strain, asCAP0167, and asCAC1766. Additional data file 9 is a table listing the primer sequences used in the Q-RT-PCR experiments.Supplementary MaterialAdditional data file 1Comparison of the present microarray study to an earlier microarray study that examined the early sporulation of C. acetobutylicum.Click here for fileAdditional data file 2COG analysis for each cluster and all the genes placed in each cluster.Click here for fileAdditional data file 3Transcriptional profiles, in terms of both intensity and differential expression, of specific gene clusters.Click here for fileAdditional data file 4Includes a brief discussion on how the genes used to construct the deduced activity plots were chosen.Click here for fileAdditional data file 5Differential expression and intensity of all annotated histidine kinases and response regulators.Click here for fileAdditional data file 6Phylogenetic tree resulting from the alignment of the σ70-related and unannotated sigma factors from ten bacterial species.Click here for fileAdditional data file 7Sequences for each asRNA construct.Click here for fileAdditional data file 8TEM images of the plasmid control strain, asCAP0167, and asCAC1766.Click here for fileAdditional data file 9Primer sequences used in the Q-RT-PCR experiments.Click here for file\n\nREFERENCES:\nNo References"
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batch_11/PMC2531111.json

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+{
+    "id": "PMC2531111",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2531111\nAUTHORS: Syed Ahmer, Rashid AM Khan, Saleem Perwaiz Iqbal\n\nABSTRACT:\nBackgroundIt has been known for a long time that use of antipsychotics, particularly atypical antipsychotics, is associated with weight gain and increase in risk of metabolic disturbances. In this study we have tried to find out if use of antipsychotics is associated with increase in weight and body mass index (BMI) in the Pakistani population.MethodsWe performed a case note review of all patients who had been prescribed antipsychotic medication at the psychiatry outpatient clinic of a tertiary care university hospital in Pakistan over a 4-year period.ResultsA total of 50% of patients had a BMI in the overweight or higher range at baseline. Patients showed a mean weight gain of 1.88 kg from baseline in 3 months and 3.29 kg in 6 months. Both of these values were statistically significant. The increase in mean BMI from baseline was 0.74 and 1.3 in 3 months and 6 months, respectively. In patients for whom we had at least one further weight measurement after baseline, 48% (39/81) showed a clinically significant weight gain.ConclusionPakistani patients are just as likely to put on weight during antipsychotic treatment as patients from other countries. Considering that this population already has a much higher prevalence of diabetes mellitus compared to the Western countries, the consequences of increased weight may be even more serious in terms of increased morbidity and mortality.\n\nBODY:\nBackgroundThe mortality rate of people suffering from schizophrenia has been estimated to be twice as high as in the general population[1]. More than two thirds of this excess mortality is due to 'natural' causes[2], with death due to cardiovascular complications being the leading cause of this excess mortality[3].The first reports of an increased risk of impaired glucose tolerance in people suffering from schizophrenia appeared in the literature several years before the first antipsychotic became available[4,5]. Soon after chlorpromazine was discovered reports suggesting an association between chlorpromazine and diabetes started appearing[6]. Since then many studies have been published firmly establishing a clear link between antipsychotics and diabetes mellitus, more with atypical than typical antipsychotics [7-10]. This led to a US Food and Drug Administration (FDA) recommendation in 2003 for including a warning about association with hyperglycaemia and diabetes on product labels for all atypical antipsychotics[9].While it is not entirely clear how antipsychotics are linked to increased risk of impaired glucose tolerance and diabetes, weight gain and obesity are major side effects of many antipsychotics [11-13]. Obesity itself leads to hypertension, type II diabetes and coronary heart disease, many of the same problems that people with schizophrenia are already at an increased risk for[12].We have not come across any research studying the association between antipsychotic use and weight gain in a Pakistani population. In this study, we have tried to find out if use of antipsychotics is associated with increase in weight and body mass index (BMI) in this population.MethodsThe study was a case note review of all patients who had been prescribed antipsychotic medication in the psychiatry outpatient clinic of the Aga Khan University Hospital (AKUH) over a 4-year period. Patients were identified using the Psychiatric Assessment System (PAS), which records the basic demographic and clinical details including the medication prescribed, of patients presenting to the psychiatry clinics at the AKUH for the first time. All patients have their height recorded on the first visit and weight on every visit.We calculated mean weight and BMI (weight in kg/height in m2) at baseline, 3 months and 6 months. A World Health Organization (WHO) expert consultation has suggested that the BMI cut-off points for determining overweight and obesity for Asian populations may be lower than Caucasian populations[14]. The consultation suggested the intervals of < 18.5, 18.5 to 23, 23 to 27.5 and ≥ 27.5, representing the categories of being underweight, increasing but acceptable risk, increased risk, and higher risk, respectively. We have used the same cut-offs in this study.An increase in weight of 7% or more compared to the baseline is considered by licensing authorities as clinically significant weight gain[15]. We calculated how many patients had achieved clinically significant weight gain at 3 months and 6 months.Statistical analyses were performed in SPSS v.15 (SPSS Inc., Chicago, IL, USA). We calculated means (with standard deviations) for quantitative variables and proportions (percentages) for categorical characteristics. We used a paired t test to determine if patients had achieved a statistically significant increase in weight and BMI from baseline. p Values < 0.05 were considered significant.ResultsWe found a total of 145 patients who had been seen at least once in the psychiatry clinic of AKUH and had been prescribed an antipsychotic medication. All of these had had their weight recorded at baseline. A total of 81 patients had at least 1 further weight measurement at least 3 months after the baseline measurement. In all, 33 patients had their weight measured at all 3 time points; baseline, 3 months and 6 months. A total of 56 people had been weighed at baseline and 3 months, and 60 people at baseline and 6 months.The baseline sociodemographic and clinical characteristics of the sample are given in Table 1.Table 1Patient demographics and clinical characteristics at baselineParameterValueAge, years median (interquartile range)31 (24–43)Gender (n = 141):Male79 (56%)Female62 (44%)Marital status (n = 138):Single75 (51%)Married52 (35.4%)Widowed7 (4.8%)Divorced3 (2%)Separated1 (0.7%)Psychiatric diagnosis (n = 145):Schizophrenia85 (57.8%)Depression21 14.3%Bipolar disorder16 (10.9%)Delusional disorder6 (4.1%)Learning disability5 (3.4%)Dementia3 (2%)Substance misuse3 (2%)Obsessive/compulsive disorder (OCD)2 (1.4%)Anorexia nervosa2 (1.4%)Attention-deficit hyperactivity disorder (ADHD)1 (0.7%)Personality disorder1 (0.7%)Antipsychotic prescribed (n = 145):Risperidone75 (51%)Olanzapine23 (15.6%)Quetiapine9 (6.1%)Aripiprazole3 (2%)Clozapine1 (0.7%)Typical antipsychotics34 (23.1%)The mean weight and BMI of the total sample at baseline, 3 months and 6 months are shown in Table 2. Among all patients for whom we could calculate BMI (n = 140) 50% (70/140) had a BMI in the overweight or higher range (> 23) at baseline, 61% at 3 months and 63% at 6 months.Table 2Mean (SD) weight and body mass index (BMI)Baseline3 months6 monthsWeight, kg63.28 (16.99)65.40 (18.01)65.79 (15.79)BMI, kg/m223.65 (5.45)25.02 (5.48)25.18 (4.93)SD, standard deviation.Patients for whom we had weight readings at baseline and 3 months (n = 56) showed a mean weight gain of 1.88 kilograms (63.51 vs 65.4 kg). This difference was statistically significant (t = -3.16, p value = 0.003). Patients for whom we had weight readings at baseline and 6 months (n = 60) showed a mean weight gain of 3.29 kilograms (62.5 vs 65.79 kg). This difference was also statistically significant (t = -2.95, p value = 0.004).The difference in mean BMI at baseline and 3 months was 0.74 (24.27 and 25.02 respectively), which was statistically significant (p = 0.002). The difference in mean BMI between baseline and 6 months was 1.3 (23.84 and 25.18 respectively) and this increase was also statistically significant (p value = 0.002)In patients for whom we had at least 1 further weight measurement after baseline, 48% (39/81) showed a clinically significant weight gain. In all, 51% (19/37) of patients on risperidone, 71% (8/11) on olanzapine and 16% (1/6) on quetiapine achieved clinically significant weight gain. However, the numbers were too small to meaningfully assess differences in the propensity of different antipsychotics to cause clinically significant weight gain.We did a secondary analysis, dividing patients into groups by psychotic disorders, (schizophrenia, delusional disorder, drug-induced psychosis) and non-psychotic disorders (all other diagnoses) but the differences between the weights of these groups were non-significant at all time points (p value 0.671 at baseline, 0.238 at 3 months and 0.645 at 6 months).A total of 91 patients were taking other psychotropic(s) besides an antipsychotic medication; 34 of these were taking SSRIs, 7 TCAs, 17 anticholinergics, 25 mood stabilisers (out of these 13 were taking valproic acid), 12 benzodiazepines, and 8 zolpidem. In all, 12 patients were taking other antidepressants including Mirtazapine (3), venlafaxine (5), and Mianserin (4).DiscussionIn this study we found that almost 50% of patients had a BMI in the overweight or higher range according to the WHO suggested cut-offs for Asian populations at the start of the study. On average patients gained about 2 kg and 3.5 kg in weight from baseline in 3 and 6 months, respectively. This correlated with a BMI increase of 0.74 in 3 months and 1.3 in 6 months. About 48% of patients for whom we had at least 1 more weight reading after 3 or 6 months achieved a clinically significant weight gain.In the study by Zipursky et al. [11] patients receiving olanzapine or haloperidol had a mean weight gain of 15.4 kg and 7.5 kg respectively. Allison et al. [12] in their systematic review reported a range of weight gain from 0.04 kg for ziprasidone to 4.45 kg for clozapine. Taylor and McAskill [13] concluded that all atypical antipsychotics, with the exception of ziprasidone (aripiprazole had not been marketed in 2000), have been associated with weight increases, with clozapine having the highest risk. The weight gain in our study was closer to the Allison than the Zipursky study. The main reason for this difference could be that in the Zipursky study patients were not recruited if they had received prior antipsychotic treatment for more than 16 cumulative weeks.The overall prevalence of diabetes mellitus in Pakistan has been reported to be between 8.6% and 13.9%, depending on the province of residence [16-18]. This is far higher than the prevalence of diabetes of 1.2 to 6.3% reported from the US [8] or around 3% reported from the UK [19]. Any drug that causes weight gain is, therefore, likely to have even more serious consequences in terms of morbidity and mortality for the Pakistani population.One of the limitations of our study was that almost all the patients had already received one or more antipsychotics for variable lengths of time before they first presented to the clinic at the AKUH. That may explain whey the weight gain in our study was not as stark as the Zipursky study[11]. Another limitation of the study is that there was no control group of patients who were not taking antipsychotic medications. This would have shed some light on how much of the weight gain might be attributable to suffering from a psychiatric illness and how much to taking of antipsychotic medications.ConclusionAntipsychotics are associated with statistically significant weight gain in the Pakistani population. This may be even more hazardous for this population as the prevalence of diabetes mellitus is already higher than many other countries. It is important that while initiating an antipsychotic medication in this patient population, psychiatrists should counsel patients about the risk of weight gain associated with antipsychotic use, the increased risk of morbidity and mortality associated with weight gain, and the lifestyle changes such as changes in dietary habits and regular exercise that the patients can adopt to counter that risk.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsSA carried out the literature review, wrote the protocol, and wrote the initial draft of the paper. RK performed data extraction and was responsible for data entry into SPSS. SPI wrote the statistical part of the protocol/paper and carried out the statistical analyses. All authors were responsible for drafting the final form of the paper and approved the manuscript.\n\nREFERENCES:\nNo References"
+}

batch_11/PMC2531112.json

@@ -0,0 +1,4 @@
+{
+    "id": "PMC2531112",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2531112\nAUTHORS: Peter H Silverstone, Robert Williams, Louis McMahon, Rosanna Fleming, Siobhan Fogarty\n\nABSTRACT:\nBackgroundBupropion HCl is a widely used antidepressant that is known to cause seizures in a dose-dependent manner. Many patients taking antidepressants will consume alcohol, even when advised not to. Previous studies have not shown any interactions between bupropion HCl and alcohol. However, there have been no previous studies examining possible changes in seizure threshold induced by a combination of alcohol and bupropion HCl.MethodsExperimentally naïve female Swiss albino mice (10 per group) received either single doses of bupropion HCl (ranging from 100 mg/kg to 120 mg/kg) or vehicle (0.9% NaCl) by intraperitoneal (IP) injection in a dose volume of 10 ml/kg, and single-dose ethanol alone (2.5 g/kg), or vehicle, 5 min prior to bupropion dosing. The presence or absence of seizures, the number of seizures, the onset, duration and the intensity of seizures were all recorded for 5 h following the administration of ethanol.ResultsThe results show that administration of IP bupropion HCl alone induced seizures in mice in a dose-dependent manner, with the 120 mg/kg dose having the largest effect. The percentage of convulsing mice were 0%, 20%, 30% and 60% in the 0 (vehicle), 100, 110, and 120 mg/kg dose groups, respectively. Pretreatment with ethanol produced a larger bupropion HCl-induced convulsive effect at all the doses (70% each at 100, 110 and 120 mg/kg) and a 10% effect in the ethanol + vehicle only group. The convulsive dose of bupropion HCl required to induce seizures in 50% of mice (CD50), was 116.72 mg/kg for bupropion HCl alone (CI: 107.95, 126.20) and 89.40 mg/kg for ethanol/bupropion HCl (CI: 64.92, 123.10).ConclusionThese results show that in mice alcohol lowers the seizure threshold for bupropion-induced seizures. Clinical implications are firstly that there may be an increased risk of seizures in patients consuming alcohol, and secondly that formulations that can release bupropion more readily in alcohol may present additional risks to patients.\n\nBODY:\nIntroductionBupropion HCl is known to cause seizures both when given at therapeutic doses or following accidental or intentional overdose in a dose-dependent manner [1-7]. It is also known that factors which include the excessive use of alcohol and sedatives, history of head trauma or prior seizure, and substance abuse, to mention a few, are associated with increased risk of bupropion-induced seizures [7]. In addition, postmarketing surveillance reports have indicated that there have been rare cases of adverse neuropsychiatric events or reduced alcohol tolerance in patients who are taking alcohol during treatment with bupropion [7]. Despite these latter reports, previous studies of the pharmacokinetic and/or pharmacodynamic interactions between alcohol and bupropion have revealed no significant pharmacodynamic interactions in animals [8], and no pharmacokinetic interactions in healthy human volunteers [9]. Furthermore, there are no studies specifically investigating the interaction between alcohol and bupropion-induced seizures in animals or man. Therefore, the objective of this study was to evaluate the effect of ethanol pretreatment on single-dose bupropion HCl-induced seizures in the Swiss albino mouse model.Materials and methodsThe study protocol and any amendment(s) or procedures involving the care and use of animals were reviewed and approved by an appropriate ethics committee following internationally approved guidelines (Charles River Laboratories Preclinical Services Inc.'s (CRM) Institutional Animal Care and Use Committee; Charles River Laboratories, Wilmington, MA, USA). During the study, the animals were maintained in a facility fully accredited by the Standards Council of Canada (SCC) and the care and use of the animals was conducted in accordance with the guidelines of the Canadian Council on Animal Care (CCAC).AnimalsExperimentally naïve female Swiss Crl: CD1 (ICR) albino mice (Mus Musculus; Charles River Canada Inc., St. Constant, Quebec, Canada) of approximately 7 weeks of age, and weighing 17.3 to 28.6 g were housed individually in stainless steel wire mesh-bottomed cages equipped with an automatic watering valve in an environmentally controlled vivarium (temperature 22 ± 3°C; relative humidity 50 ± 20%) with a 12-h light/dark cycle. All animals were acclimated to their cages and to the light/dark cycle for 3 days before the initiation of treatment. In addition, all animals had free access ad libitum to a standard certified pelleted commercial laboratory diet (PMI Certified Rodent Diet 5002; PMI Nutrition International Inc., St Louis, MO, USA) and tap water except during designated procedures. Animals were randomly assigned to 8 treatment groups of 10 mice per group, using a computer-generated randomisation scheme, ensuring stratification by body weights. Four groups were pretreated with ethanol followed by treatment with increasing doses of bupropion HCl as follows: group 1, ethanol 2.5 g/kg + 0 mg/kg (vehicle); group 2, ethanol 2.5 g/kg + 100 mg/kg; group 3, ethanol 2.5 g/kg + 110 mg/kg; and group 4, ethanol 2.5 g/kg + 120 mg/kg. The other four groups were only treated with the same increasing doses of bupropion HCl as follows: group 5, 0 mg/kg (vehicle only); group 6, 100 mg/kg; group 7, 110 mg/kg; and group 8, 120 mg/kg. The doses of bupropion HCl 100 to 120 mg/kg selected for this study are higher than the low dose of 12.5 mg/kg used in a previous study [8] because more recent studies have revealed that bupropion HCl at low doses of 15 to 30 mg/kg does not induce seizures but protects albino mice against seizures induced by maximal electroshock (anticonvulsant), and at high doses of 100 to 160 mg/kg is proconvulsant in the mice [10]. Animals in poor health or at the extremes of the prespecified body weight range (18 to 30 g) were not assigned to treatment groups and unassigned animals were released from the study.DrugsBupropion HCl was obtained from Biovail Corporation, Steinbach, Manitoba, Canada, in white powder form. The dose formulations of bupropion HCl were prepared on each day. The appropriate amount of bupropion HCl was weighed and dissolved in an appropriate amount of 0.9% NaCl and then vortexed until a solution was obtained. On each day of treatment, the single doses of bupropion HCl dose were administered by intraperitoneal (IP) injection in a dose volume of 10 ml/kg and dose concentrations of 0, 10, 11, and 12 mg/ml for the 0, 100, 110, and 120 mg/kg doses. The actual dose administered was based on the most recent body weight of each animal. In the applicable treatment groups (groups 1 to 4), each animal was pretreated with ethanol in a dose volume of 10 ml/kg 5 min prior to bupropion dosing. Ethanol was obtained in liquid form from Les Alcools de Commerce Inc., Montreal, Quebec, Canada. Ethanol 2.5 g/kg was administered as a dose volume of 10 ml/kg, and a dose concentration of 0.25 g/ml. Vehicle was 0.9% sodium chloride (NaCl) for injection USP and was obtained from Baxter Healthcare Corporation, Deerfield, IL, USA.Study procedureAll animals were examined twice daily for mortality and signs of ill health or reaction to treatment, except on the days of arrival and necropsy when they were examined only once. After the acclimation period and randomisation, on the day prior to the initiation of treatment, all animals were weighed and the individual body weights were used for dose volume calculation. Treatment was then initiated and lasted for 4 consecutive days with equal numbers of animals from each group dosed on each day. On the days of treatment, approximately 5 min prior to bupropion HCl or vehicle dosing, animals in groups 1 to 4 were pretreated with a single dose of ethanol 2.5 g/kg IP in a dose volume of 10 ml/kg. These animals then received the assigned dose of bupropion HCl or vehicle IP. Animals in groups 5 to 8 were not pretreated with ethanol but received their assigned dose of bupropion HCl or vehicle by the IP route. Thereafter, the animals were placed in clear perspex observation boxes containing a foam base for padding and observed for the occurrence of seizures for 5 h, followed by a 5 min assessment at 24 h post dose. The presence or absence of seizures, the number of seizures, the onset, duration and intensity of seizures were all recorded. The intensity of each convulsion was graded using Charles River Laboratories, Inc.'s grading system of mild: head and tail slightly extended and little jerking; moderate: head and tail fully extended and some jerking; or severe: head and tail fully extended and strong jerking. In addition, the presence or absence of ataxic gait, paralysis, and catatonic episodes (without a grading of the intensity or number) were recorded over each 15 min observation period. Any animal that had a single episode of severe seizure lasting longer than 1 min or any animal displaying greater than 40 separate episodes of severe seizures over a 1-h period was sacrificed for humane reasons. At the end of the 5-h observation period, all animals were returned to their home cages, and as deemed necessary, additional bedding, food (on cage floor) and water bottles were provided if an animal was still showing adverse effects from the administration of study drugs.Assessment of convulsant activityThe primary outcome variable was the percentage of mice that had seizures. This was the number of animals with seizures (mild, moderate or severe) divided by the total number of animals in each group multiplied by 100. In addition, the convulsive dose of bupropion HCl required to induce seizures in 50% of mice (CD50), was calculated for the dose-response curves for bupropion HCl treatment alone and the ethanol/bupropion HCl treatment. The secondary outcome variables were the mean (SD) seizures per mouse in each group, and the duration of seizures.Data presentation and statistical analysisData was summarised and presented in tables by treatment groups for the primary outcome variable, the percentage of convulsing mice, and the two secondary outcome variables, the mean (SD) seizures per mouse in each group, and the duration of seizures. The CD50 values were calculated using the PROBIT procedure in SAS (SAS Inc., Cary, NC, USA). The 95% confidence limits for CD50 were calculated according to the method of Litchfield and Wilcoxon [11]. A total of 10 mice per group (total of 40 animals) were used to calculate the CD50 for the bupropion alone treatments, and 39 animals for the CD50 for the ethanol/bupropion HCl treatments. The number of seizures per mouse was analysed using analysis of variance (ANOVA) on the rank-transformed values, with presence of ethanol (yes/no), bupropion dose, and presence of ethanol-by-bupropion dose interaction as fixed effects in the model. p Values of ≤ 0.05 were considered statistically significant.ResultsIn all groups, except the group treated with vehicle only (group 5), a convulsive effect was observed following the administration of bupropion HCl and/or ethanol. The onset of convulsion was about 9 min following the administration of single doses of bupropion HCl, however, this was highly variable between animals in the same group and across the dose levels for the bupropion HCl alone and ethanol/bupropion HCl treatments. The intensity of the seizures observed following bupropion HCl alone treatment were only mild and moderate (Table 1). Following ethanol pretreatment, overall, there was an increase in the intensity of the bupropion HCl-induced seizures at all the doses. In the 100 mg/kg dose group (group 5), there were marked increases in the number of mild, moderate and severe seizures. In the 110 and 120 mg/kg dose groups, there was a redistribution of the intensity of the seizures resulting in reductions in the mild seizures but a fivefold and twofold increase, respectively, in the moderate seizures (Table 1).Table 1Effect of ethanol pretreatment on bupropion HCI-induced convulsions: intensity of convulsionsDose (mg/kg), n = 10 per groupIntensity of convulsionsMildModerateSevereBUPET + BUPBUPET + BUPBUPET + BUP0 (V or ET +V)010100100137165071102110150012019172400n = 10 mice per group for bupropion HCl alone and ethanol + bupropion HCl treatment groups.BUP, bupropion HCl; ET, ethanol; V, vehicle or 0.9% sodium chloride (NaCl).There were no deaths in the study. One animal treated with ethanol/bupropion HCl 110 mg/kg had excessive convulsions and was therefore euthanised for humane reasons. A variety of clinical signs were observed in the mice following the administration of bupropion HCl, some of which include paralysis, ataxic gait, catatonia, increased respiratory rate, twitching, tremors, increased activity, decreased activity, partially closed eyes, etc. Clinical signs were not dose dependent and pretreatment with ethanol had no effect on the signs observed.Percentage of convulsing miceAdministration of single doses of IP bupropion HCl alone induced seizures in mice in a dose-dependent manner with the 120 mg/kg dose showing the largest effect. The percentage of convulsing mice were 0%, 20%, 30% and 60% in the 0 (vehicle only = 0.9% NaCl), 100, 110, and 120 mg/kg dose groups, respectively (Table 2 and Figure 1). Pretreatment with ethanol produced a larger bupropion HCl-induced convulsive effect at all the doses including the ethanol + vehicle only group. There was a marked increase in the percentage of convulsing mice (70% of convulsing mice) at the ethanol/bupropion HCl 100 mg/kg dose, compared to bupropion HCl alone treatment, which was maintained at the ethanol/bupropion HCl 110 and 120 mg/kg doses, resulting in a flat dose-response curve (Table 2 and Figure 1). Ethanol/vehicle (group 1) treatment induced a 10% incidence of seizures.Figure 1Dose-response curves of the percentage of convulsing mice following the administration of bupropion HCl alone (closed circles) and the effect of ethanol pretreatment on bupropion HCl-induced seizures (open circles) in the Swiss albino mice. The 50% convulsing dose (CD50) values, the convulsant doses of bupropion HCl required to induce seizures in 50% of mice were 116.72 (CI: 107.95, 126.20) and 89.40 (CI: 64.92, 123.10) mg/kg for the dose-response curves for bupropion alone and ET + bupropion HCl, respectively. Doses of bupropion HCl administered intraperitoneally (IP) were 0 (vehicle or ET + vehicle only), 100, 110, and 120 mg/kg. Ethanol pretreatment was with 2.5 g/kg IP 5 min prior to administration of bupropion HCl. Each data point is the percentage of convulsing mice in n = 10 mice. ET, ethanol + vehicle; S, vehicle (0.9% NaCl).Table 2Effect of ethanol pretreatment on bupropion HCI-induced convulsions: percentage of convulsing miceDose (mg/kg), n = 10 per groupNo. of convulsing micePercentage of convulsing miceBupropion HClET + Bupropion HClBupropion HClET + Bupropion HCl0 (vehicle or ET+vehicle)010%10%1002720%70%1103730%70%1206760%70%n = 10 mice per group for bupropion HCl alone and ethanol + bupropion HCl treatment groups.ET, ethanol; vehicle, 0.9% sodium chloride (NaCl).The CD50 or convulsive dose50, the convulsive doses of bupropion HCl required to induce seizures in 50% of mice, were 116.72 (CI: 107.95, 126.20) and 89.40 (CI: 64.92, 123.10) mg/kg for the dose-response curves for bupropion alone and ethanol/bupropion HCl treatments, respectively (Figure 1). The CD50 of 116.72 (CI: 107.95, 126.20) mg/kg for bupropion HCl alone treatment is similar to the value of 119.7 (CI: 104.1, 137.6) mg/kg reported previously for IP bupropion HCl in Swiss mice [10].Mean convulsions per mouseThe analysis of variance results showed a significant overall effect of ethanol pretreatment and bupropion dose on the number of bupropion HCl-induced seizures, and a borderline significant overall ethanol-bupropion interaction effect at the p ≤ 0.10 level (Table 3). Single-dose bupropion HCl alone treatment induced a dose-dependent increase in the mean (SD) seizures per mouse from 0 in the vehicle only-treated group (bupropion HCl 0 mg/kg) to 2.20 (4.49) seizures per mouse in the 110 mg/kg dose group, which was maintained at the 120 mg/kg dose group (mean (SD) convulsions per mouse = 2.10 (1.97)). Pretreatment with ethanol markedly and significantly increased the mean (SD) seizures per mouse compared to bupropion HCl alone treatment only in the 100 mg/kg dose group (ethanol/bupropion HCl = 10.90 (17.28); bupropion HCl alone = 0.20 (0.42); p = 0.0019). There were no statistically significant differences between the mean (SD) seizures per mouse obtained for ethanol/bupropion HCl versus bupropion alone treatments for the 0, 110 and 120 mg/kg dose groups (Table 3).Table 3Effect of ethanol pretreatment on bupropion HCI-induced convulsions: mean standard deviation (SD) convulsions per mouseDose (mg/kg), n = 10 per groupTotal no. of convulsionsMean (SD) convulsions per mouseBUPET + BUPBUPET + BUPp Value0 (V or ET +V)020.00 (0.00)0.20 (0.63)0.1027*10021090.20 (0.42)10.90 (7.28)†11022152.20 (4.49)1.50 (1.72)12021212.10 (1.97)2.10 (3.35)n = 10 mice per group for bupropion HCl alone and ethanol + bupropion HCl treatment groups.*p Value for overall ethanol-bupropion interaction effect (ethanol effect, overall p = 0.0183; bupropion dose effect, overall p = 0.0007).†p = 0.0019 for pairwise comparison with corresponding mean value for bupropion alone treatment.BUP, bupropion HCl; ET, ethanol; SD, standard deviation; V, vehicle or 0.9% sodium chloride (NaCl).Duration of convulsionsAdministration of single doses of bupropion HCl alone induced only short and medium duration seizures. The number of short seizures increased with dose to a maximum of 22 at the 110 mg/kg dose with a slight decrease to 18 at the 120 mg/kg dose (Table 4). In contrast, pretreatment with ethanol increased the total numbers of bupropion HCl-induced short and medium seizures, as well as caused long seizures. In addition, the number of short, medium and long seizures was markedly highest at the 100 mg/kg dose followed by a marked reduction at the 110 mg/kg dose and a further reduction at the 120 mg/kg dose only for the medium and long seizures (Table 4).Table 4Effect of ethanol pretreatment on bupropion HCI-induced convulsions: duration of convulsionsDose (mg/kg),n = 10 per  groupDuration of convulsionsNo. of short convulsions (0 to 10 s)No. of mediumconvulsions  (11 to 30 s)No. of long convulsions (≥ 31 s)BUPET + BUPBUPET + BUPBUPET + BUP0 (V or ET +V)010100100178117014110226030612018173103n = 10 mice per group for bupropion HCl alone and ethanol + bupropion HCl treatment groups.BUP, bupropion HCl; ET, ethanol; V, vehicle or 0.9% sodium chloride (NaCl).DiscussionThe pharmacokinetic and pharmacodynamic interactions of ethanol with antidepressant drugs are well known [12-17]. Interactions between ethanol and psychotropic drugs could be additive, synergistic (potentiation) or antagonistic [15]. Even though there are published reports of animal [8] and human [9,18] studies investigating the pharmacokinetic and/or pharmacodynamic interactions between alcohol and bupropion, there are no published studies precisely evaluating the effects of alcohol on the convulsive liability of bupropion. This study was therefore designed to investigate the effect of ethanol pretreatment on single-dose bupropion HCl-induced seizures in the Swiss albino mice. The results of the primary outcome variable showed that bupropion HCl alone treatment in the dosage range 0 to 120 mg/kg was associated with a dose-dependent increase in the percentage of mice with bupropion HCl-induced seizures. This finding is consistent with previous reports that indicate bupropion induces seizures in a dose-dependent manner in animals [10,19] and humans [2,3,7]. Pretreatment with ethanol resulted in markedly increased percentage of mice with bupropion HCl-induced seizures at the 100 mg/kg dose, which was maintained at the 110 and 120 mg/kg doses. The latter results are consistent with a 3.5-, 2.3- and 1.2-fold increase in the percentage of convulsing mice at the 100, 110 and 120 mg/kg doses, respectively, following ethanol pretreatment. In addition, ethanol pretreatment resulted in a flat dose-response within the dosage range of 100 to 120 mg/kg studied. The CD50 for bupropion HCl alone treatment, a well known index of convulsive liability, of 116.72 (CI: 107.95, 126.20) mg/kg is similar to the value of 119.7 (CI: 104.1, 137.6) mg/kg reported previously for bupropion HCl in Swiss mice [10], and confirms the validity of this animal model. Pretreatment with ethanol resulted in a 23% reduction in the CD50 value for bupropion HCl-induced seizures.The results of the secondary outcome variables were generally consistent with the results of the primary outcome variable. Bupropion HCl alone treatment induced a dose-dependent increase in the mean seizures per mouse up to the 110 mg/kg dose, which was maintained at the 120 mg/kg dose. Ethanol pretreatment resulted in a marked and statistically significant 54-fold increase in bupropion HCl-induced mean seizures per mouse only at the 100 mg/kg dose. There were no significant differences in bupropion HCl-induced mean seizures per mouse at the 110 and 120 mg/kg doses following ethanol pretreatment. With respect to the duration of seizures, bupropion HCl alone treatment only induced short and medium duration seizures, which when combined was dose dependent up to the 110 mg/kg dose. Ethanol pretreatment increased the duration of the seizures overall, resulting in more episodes of short, medium, and long duration bupropion HCl-induced seizures, but particularly in the 100 mg/kg dose group.The results of this study are in conflict with the results of previous studies that reported no pharmacodynamic interactions between alcohol and bupropion in mice [8], and no pharmacokinetic interactions in normal healthy volunteers [9]. The reason for the discrepant previous results may be because those studies of the pharmacokinetic and pharmacodynamic interactions between alcohol and bupropion in normal healthy volunteers [9,18] used a low dose of bupropion (100 mg, approximately 1.5 mg/kg) that is unlikely to be associated with the occurrence of seizures since bupropion-induced seizures are dose dependent. Similarly, a previous study [8] investigating the interactive effect of combined treatment with alcohol and bupropion in adult albino mice utilised a low dose of bupropion (12.5 mg/kg IP) which is much lower than the convulsive doses of 100 to 160 mg/kg IP, with a CD50 of 119.7 (CI: 104.1, 137.6) mg/kg and CD97 of 156.7 mg/kg, that were subsequently reported for bupropion in mice by other investigators [10]. In addition, lower doses of bupropion (15 to 30 and 5 to 10 mg/kg, respectively), which did not induce seizures, have been reported to protect against seizures evoked by maximal electroshock [10] and nicotine [20] in mice. However, one group has reported that the combination of bupropion with alcohol abolished the impairment in auditory vigilance and mental slowness observed following the administration of alcohol alone in normal healthy volunteers (a pharmacodynamic interaction) even though they used a low dose of bupropion (100 mg) and found no pharmacokinetic interaction [18].The mechanism of bupropion HCl-induced seizures is unknown [21,22]. Similarly, the mechanism for the synergistic interaction reported here between ethanol and bupropion HCl is also unknown. This interaction is unlikely to be solely due to pharmacokinetic reasons since a previous crossover study that investigated the interactions between alcohol and bupropion found no such interactions [9]. This previous study, also in normal healthy human volunteers, examined the effect of administration of oral bupropion HCl 100 mg followed by the administration of ethanol found no changes in the pharmacokinetics of bupropion, and vice versa [9].The observed interaction between ethanol and bupropion reported in the present study has potential clinical implications. It has been recognised that the seizure risk of bupropion is increased in subjects undergoing abrupt withdrawal from alcohol [3,4], hence, bupropion administration is contraindicated in such patients [7]. However, the more recent although rare postmarketing reports of adverse neuropsychiatric events or reduced alcohol tolerance in patients who are drinking alcohol during treatment with bupropion [7], suggests that there is an interaction between alcohol and bupropion following coadministration, consistent with the findings of this study. Consequently, patients should be cautioned to not consume alcohol with bupropion. Nonetheless, there is good evidence that many patients on bupropion, as well as other anti-depressants, continue to use alcohol [23].In conclusion, the results of this study demonstrate that ethanol pretreatment followed by single-dose IP bupropion HCl resulted in an increase in the number and percentage of convulsing mice, mean seizures per mouse, the intensity, and the duration of the seizures. Following ethanol pretreatment, the CD50 for bupropion HCl alone treatment was reduced from 116.7 to 89.0 mg/kg, representing a 23% reduction. The dose-related increase in the percentage of convulsing mice and mean seizures per mouse is consistent with previous reports that bupropion-induced seizures are dose dependent in animals and humans. The observed pharmacodynamic interaction between ethanol and bupropion-induced seizures in this study is novel and the mechanism is unknown. However, it has potential clinical implications for the prescribing of bupropion. It also implies that caution should be used when bupropion is prescribed to patients either using alcohol or at high risk of doing so.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsThe study was conceived by PHS and RW, was designed by LM and SF who were also involved in data acquisition, the first draft of the paper was by PHS, it was carried out in part by LM, and the statistical analysis was by RF. Funding for the conduct of this study and the manuscript preparation was provided by Biovail Laboratories International SRL.\n\nREFERENCES:\nNo References"
+}

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+{
+    "id": "PMC2531117",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2531117\nAUTHORS: Sara Borin, Elena Crotti, Francesca Mapelli, Isabella Tamagnini, Cesare Corselli, Daniele Daffonchio\n\nABSTRACT:\nBackgroundExtracellular dissolved DNA has been demonstrated to be present in many terrestrial and aquatic environments, actively secreted, or released by decaying cells. Free DNA has the genetic potential to be acquired by living competent cells by horizontal gene transfer mediated by natural transformation. The aim of this work is to study the persistence of extracellular DNA and its biological transforming activity in extreme environments like the deep hypersaline anoxic lakes of the Mediterranean Sea. The brine lakes are separated from the upper seawater by a steep chemocline inhabited by stratified prokaryotic networks, where cells sinking through the depth profile encounter increasing salinity values and osmotic stress.ResultsSeven strains belonging to different taxonomic groups isolated from the seawater-brine interface of four hypersaline lakes were grown at medium salinity and then incubated in the brines. The osmotic stress induced the death of all the inoculated cells in variable time periods, between 2 hours and 144 days, depending on the type of brine rather than the taxonomic group of the strains, i.e. Bacillaceae or gamma-proteobacteria. The Discovery lake confirmed to be the most aggressive environment toward living cells. In all the brines and in deep seawater dissolved plasmid DNA was substantially preserved for a period of 32 days in axenic conditions. L'Atalante and Bannock brines induced a decrease of the supercoiled form up to 70 and 40% respectively; in the other brines only minor changes in plasmid conformation were observed. Plasmid DNA after incubation in the brines maintained the capacity to transform naturally competent cells of Acinetobacter baylii strain BD413.ConclusionFree dissolved DNA is likely to be released by the lysis of cells induced by osmotic stress in the deep hypersaline anoxic lakes. Naked DNA was demonstrated to be preserved and biologically active in these extreme environments, and hence could constitute a genetic reservoir of traits acquirable by horizontal gene transfer.\n\nBODY:\nBackgroundFree extracellular DNA has been retrieved in many environments, both aquatic and terrestrial [1]. Naked DNA is actively excreted by growing cells, depending on biotic and abiotic factors [2-4], or is passively released in the environment by decaying cells. In hypersaline systems the water availability is reduced with the consequence of inferring water stress to cells. The osmotic stress in particular affects cell turgor and membrane integrity, leading to death by osmotic lysis of cells not adapted to hypersaline conditions [5], and in turn to the release of the cellular content, including nucleic acids.Free dissolved DNA is used by microorganisms as source of N, P and C [6,7], and in the deep-sea environment has been hypothesised to constitute a key trophic resource, substantially contributing to P cycling [7]. Other than a nutrient source, dissolved DNA may have a genetic function as a source of genes acquirable by natural transformation. In terrestrial and aquatic environments several bacterial strains have been discovered to be naturally competent, i.e. to have the capacity to acquire exogenous naked DNA [4]. The acquisition of new genetic traits by horizontal gene transfer can constitute an evolutionary strategy for the selection of natural microbial communities [8]. In harsh and stressful conditions, in particular, gene exchange and rearrangements are estimated to increase in order to promote genome plasticity, increasing DNA repairing rates and evolutionary adaptation mechanisms [9,10].In natural ecosystems the majority of extracellular DNA is converted in deoxyribose, inorganic orthophosphate, purines and pyrimidines by the enzymatic hydrolytic action of nucleases, present in most of the microbial habitats [11,12]. Besides the biological degradation, DNA is a chemically unstable molecule that decays spontaneously mainly through hydrolysis and oxidation [13]. Several physical and chemical factors can moreover compromise the integrity of naked DNA molecules once they are released by cells. Once free in the environment, DNA fragments are no longer preserved by cellular DNA repair mechanisms and, even if not severely degraded, they accumulate environmentally inflicted damages. Despite all these factors, extracellular DNA has been demonstrated to be preserved in soil, sediments, freshwater and seawater for different time periods [1], and even geologically ancient DNA has been retrieved from fossil materials [14,15]. Dell'Anno and Danovaro [7] estimated that the deep-sea sediments constitute the largest reservoir of extracellular DNA in the Earth's oceans, with 0.50 ± 0,22 Gt of extracellular DNA contained in the first 10 cm of sediments, and its residence time, resulted by the balance of release and degradation, is 9.5 years. In particular, in the sediments underlying the deep anoxic hypersaline lake l'Atalante Danovaro et al. [16] retrieved the highest concentration of extracellular DNA reported in a natural environment.In hypersaline environments salt has been shown to have a stabilising effect on nucleic acids, protecting biological macromolecules against heat degradation [17,18]. On the other side, the reduced water activity of a salty environment has consequences on DNA conformation. The reduction of the hydration of the DNA molecules decreases the stabilisation of the structure that in high water activity environments is conferred by weakly bound water molecules [19].The aim of this work is to study the persistence of extracellular DNA and its potential for gene exchange in extreme environments characterised by hypersaline and anoxic conditions. The deep hypersaline anoxic lakes of the Eastern Mediterranean Sea are unique deep-sea habitats originated from the dissolution of buried salt deposits emerging at the topography due to the strong faulting activity of the area. They are characterised by a salinity above 30%, absence of light, elevated pressure, variable pH values and ionic compositions. The sharp density difference between brines and normal sea water acts as a barrier, avoiding oxygen exchange, therefore the brines become oxygen-free and rich in hydrogen sulphide. Despite these harsh conditions, the brines that fill the lakes contain highly adapted active microbial communities [20]. The brines are separated from the upper seawater by a steep interface layer with salinity values ranging from seawater to the brine physio-chemical parameters. This layer is an enrichment phase for complex microbial networks rich in taxonomical and functional biodiversity that are stratified along the depth and salinity profile [21].Results and discussionSurvival in the brines of bacteria isolated from the seawater-brine interfaceThe interface between deep sea-water and the hypersaline anoxic brines is a thin layer of few meters over the brines that hosts high bacterial density and diversity [21]. Bacteria sinking through the interface encounter conditions of increasing salinity and, as a consequence, increasing osmotic stress. Seven strains have been selected, based on the following criteria: i) isolation from the seawater-brine interface of the four hypersaline lakes, L'Atalante, Bannock, Discovery, Urania, that have brines with very different chemical composition [20] and ii) belonging to different taxonomic groups, i.e. gamma-proteobacteria and high G+C sporeforming bacteria of the family Bacillaceae (table 1). The seven strains have been isolated from the less saline layers of the hypersaline basins, i.e. the seawater-brine interface, and do not exhibit halophilic features, being able to grow in absence of salt. They are moderately halotolerant, since all of them tolerate up to 10–12% of NaCl in the growth medium (table 1). Simulating a sink in the lower hypersaline layers of the lakes, the cells of the strains isolated from the different lakes were grown on medium with intermediate salinity (5%) and then incubated in the corresponding hypersaline brines. Figure 1(A1, B1) shows the dramatic decrease in cell viability over time of contact with the brines. All the strains are subjected to a strong stress immediately after the exposure to the brines, since the number of the viable cells decreased by 5–11 orders of magnitude in the first minute. The stress is osmotic and leads to cell lysis rather than just a loss of viability, as demonstrated by the parallel decrease in optical density of the suspension (figure 1, A2, B2). The majority of the cells that were not completely lysed, showed nevertheless damaged membranes after 10 minutes of incubation in the brines, as demonstrated by their staining with propidium iodide (figure 2).Table 1Strains used in the work, taxonomic identification and salinity toleranceStrain namelake of isolation1)Medium of isolationclosest relativehomology %2)NaCl tolerance (%)minmax6AL'AtalanteDSMZ-2463)Alteromonas marina9801011AL'AtalanteDSMZ-372Halobacillus trueperi10001012BBannockMarine Broth (Difco 2216)3)Bacillus licheniformis9801218BBannockDSMZ-2463)Halomonas meridiana990105DDiscoveryDSMZ-2463)Alteromonas macleodii9901011DDiscoveryDSMZ-246Bacillus firmus9901013UUraniaMarine Broth (Difco 2216)Halomonas meridiana990101) this is also the brine in which the strain has been incubated for the survival experiments2) the strains have been identified based on the percentage homology on the 16S rRNA sequence with the sequences contained in public databases3) medium diluted 1:10 with sterile seawaterFigure 1Survival of bacterial cells in the brines. Figure 1A1, 1B1: time series plate count quantification of different strains incubated in the brine of isolation, i.e. L'Atalante, Bannock, Urania (A1) and Discovery (B1). Note that the x-axis scale in B1 is in minutes, while in A1 is in days. Figure 1A2, 1B2: time series measurement of the optical density (OD600) of the strains incubated in the brines of L'Atalante, Bannock, Urania (A2) and Discovery (B2). Error bars are within the range of 0.3–71% of each value.Figure 2Epifluorescence microscopy visualization of the strains B. licheniformis 12B (A) and H. meridiana 18B (B) after 10 minutes of incubation in the Bannock brines, stained with Propidium iodide (red) and SybrGreen (green). A1, B1: visualisation at Propidium iodide excitation/emission wavelength (494/617 nm) of the cells with damaged cell wall. A2, B2: visualisation at SybrGreen I excitation/emission wavelength (494/519 nm) of the totality of the cells.The rate of viability loss seems to depend on the type of brine rather than type of microorganism. In the Discovery brine both the strains tested, Bacillus firmus 11D and Alteromonas macleodii 5D, did not show any remnant living cell after only 2 and 24 hours of incubation respectively. When compared with the other 3 brines, the Discovery one is indeed characterised by the most extreme composition, with a concentration of MgCl2 that reaches 5 M [20], and has been shown to be highly hostile toward life [22]. In the other brines all the strains exhibited longer survival times, demonstrating viable cells up to 30–93 days, depending on the strains. Halobacillus trueperi 11A was the most resistant of all the strains tested, showing the complete disappearance of viable cells after 144 days of incubation in L'Atalante brines. Further experiments of cross inoculations would confirm whether the various degrees of lethal effect of the brines depend only by their different composition, or could be related to the peculiar physiological features of the microbes colonising each lake.Based on these results it is possible to hypothesise that, as a consequence of the osmotic stress encountered during sinking through the depth profile of the basins, cells not adapted to hypersalinity decay releasing their cellular content, including nucleic acids, during osmotic lysis. The following part of the work aims to understand the fate of naked DNA once released from the decaying cells.Survival in the brines of plasmid DNA and transformation potentialFigure 3A indicates the gel electrophoresis pictures showing the fate of the plasmid DNA during a 32 day-long incubation, whereas Figure 3B shows the relative quantification of the main plasmid bands as a mean of different experiments. In all the incubation experiments the DNA proved to be highly preserved, and the total plasmid quantity did not show remarkable degradation for the first 15 days of incubation (p < 0.01 in L'Atalante, Urania, Discovery brines). This result confirms previous findings by DeFlaun and Paul [23] who performed short term experiments of 36 hours incubation using sterile seawater. In non sterile conditions extracellular DNA is degraded in few hours both in seawater and in freshwater [23,24], due to enzymatic DNA degradation [1]. In this work the preservation of dissolved DNA has been studied in 0.22 μm pore size filtered systems, in order to selectively investigate the effects of the four anoxic brines with different ionic composition without the interference of cells that are retained on the filter. For the first 3 days of incubation brines or seawater did not show any apparent effect on DNA. After this period in the L'Atalante brine the supercoiled conformation of the plasmid (CCC form) decreased up to 70 ± 15%. The Discovery brine, which was the most aggressive toward living cells, induced a decrease up to 43 ± 25% of the CCC form between 18 and 32 days of incubation. The exposure to Urania and Bannock brines as well as the seawater did not severely affect the total quantity of DNA over the 32 days of the experiment, but mainly affected the conformation of the plasmid molecule. Except for incubation in L'Atalante brines, the CCC form decrease was minor, between 0 and 29 ± 16%, while the other forms increased in variable percentages when compared with the respective bands at the beginning of the experiment (figure 3). The incubation of pZR80(gfp) plasmid in seawater or Urania brine lead to the appearance after 8 days of a DNA band with higher electrophoretic mobility than the supercoiled form, that could be attributed to the linear plasmid. The results showed that seawater or hypersaline brines in anoxic conditions in the absence of cells and suspended material retained on 0.22 μm filters had a partial, in L'Atalante and Discovery brines, or negligible, in the other cases, degradative effect on the overall DNA molecules. This could be due to a nicking effect on the plasmid DNA molecules, leading to the opening of the supercoiled form toward more relaxed conformations. These high values of DNA preservation confirm previous findings in sediments underlying the brines of the L'Atalante lake, which reported high number of spores [25], exceptionally high concentrations of extracellular DNA [16], and in general a high level of organic matter preservation [26].Figure 3Stability of plasmid DNA in the brines and seawater. A: gel electrophoresis of pZR80(gfp) incubated for increasing periods of time in brines and seawater. B: relative quantification of the different plasmid conformations, OC (open circular), linear, CCC (covalently closed circular).To investigate the biological effect of the degradation or changes in conformation induced by brines or seawater, we tested the efficiency of the rescued plasmids in transformation. Plasmid pZR80(gfp) recovered after incubation in seawater and brines was used to transform naturally competent cells of A. baylii BD413, and the efficiency of transformation was calculated. To exclude that differences in transformation efficiency were due to differences in quantity of the donor DNA rather than in its quality, we estimated the minimum quantity of donor DNA that did not affect the transformation efficiency. The results demonstrated that applying between 20 and 50 ng of donor DNA per transformation assay did not significantly alter the transformation frequency (p < 0.03), giving an average of 1.9 ± 0.4 × 10-3 transformants/total cells (figure 4). Based on these data, the transformation efficiency of pZR80(gfp) incubated in seawater or brines was calculated using equal concentrations of 25 ng of donor DNA per assay. The results (figure 5) showed that the incubation in all the four brines or in seawater did not affect the biological activity of extracellular dissolved plasmid DNA. pZR80(gfp) maintained similar values of transformation frequency for 32 days, with an average value of 5.6 ± 3.1 10-4 transformants/total cells. Transformation frequency was on average lower than that calculated using pure plasmid extracts, probably due to incomplete desalting of plasmid preparations recovered from seawater and brines. Brines of the Urania basin that showed negligible degrading effects on dissolved DNA (figure 3), induced nevertheless a significant (p = 0.037) increase in transformation frequency after 22 days of incubation from 4.9 ± 0.3 × 10-4 to 1.2 ± 0.2 × 10-3. This result could be due to a kind of effect at molecular level induced by the brines on the dissolved DNA, that was not visible by agarose gel electrophoresis and should be further explored. The dependence of the frequency of transformation upon the topological form of the plasmid has been described [27].Figure 4Frequency of transformation of naturally competent cells of A. baylii BD413 with different quantities of plasmid pZR80(gfp).Figure 5Frequency of transformation of naturally competent cells of A. baylii BD413 with plasmid pZR80(gfp) incubated for different time periods in brines and seawater.The integrity of the genetic information acquired by horizontal gene transfer was confirmed by evaluating the expression of the gfp gene which codifies for a green fluorescent protein that conferred fluorescent phenotype to the transformants (data not shown).ConclusionAt the interface between deep seawater and brine in the hypersaline anoxic lakes of the Eastern Mediterranean Sea bacterial communities are sharply stratified according to the increasing levels of salinity. The process of particulate sinking throughout the depth profile along the interface could expose non-adapted bacteria to higher salinity, with consequent osmotic stress that induces the lysis of the cells thereby releasing nucleic acids. With this work we demonstrated that this process is likely to occur in brines with different chemical composition, the released DNA is preserved biologically active in these extreme environments as in seawater, and the preserved DNA retains its transforming potential. Even though the role 0.22 μm filterable agents like DNAses, viruses or ultramicrobacteria should be further evaluated to estimate biological degradation of extracellular DNA, and the presence of naturally competent bacteria should be demonstrated, this work gives a first insight on the potential role of dissolved extracellular DNA in hypersaline environments to constitute a reservoir of genetic information that can be acquired by horizontal gene transfer mediated by natural transformation.MethodsBrines and bacterial strainsBrines used in this study were recovered in 2003 from the deep submarine lakes Urania, Bannock, Discovery, L'Atalante located in the Eastern Mediterranean Sea, during a cruise of the R/V Urania [20]. Brines were sterilised by filtration on 0.22 μm pore size filters immediately after recovery, and then filtered a second time before the survival/preservation experiments.Strains used in the work were isolated from the seawater/brine interface of each basin [21] and maintained in Plate Count Broth (PCB) (Difco, Milan, Italy) supplemented with 5% NaCl (PCB5%). The strains have been identified by sequencing of the PCR-amplified 16S rRNA gene and comparison of the sequence with public databases [21]. The list of the strains and the isolation media are reported in table 1.Survival of bacterial cells in the brinesCells were harvested by centrifugation from 10 ml of overnight cultures incubated at 28°C in the medium PCB5%. Cells were washed with sterile NaCl solution (5%), and then resuspended in 1 ml of the same solution. One hundred μl of the bacterial suspension were inoculated in 10 ml of filter sterilised brine and incubated at 15°C, the in situ temperature of the hypersaline anoxic lakes. At defined time intervals, up to 140 days (figure 1, A1, B1), 100 μl of the incubated suspension was collected, immediately subjected to serial dilutions in NaCl solution (5%) and plate counted on agarised PCB5% in triplicate. At the same time intervals (figure 1, A2, B2) the optical density at 600 nm wavelength (OD600) of an aliquot of the suspension was analysed in a spectrophotometer (Beckman, DU640).Preservation of plasmid DNA in the brinesTo simulate the release of DNA from the decaying cells, known amounts of purified plasmid pZR80(gfp) were incubated in the filtered sterile brines of the four basins or, as a control, in deep-sea water. The incubations have been carried out simulating the in situ conditions, at 15°C in the dark and in the absence of oxygen. At selected time intervals triplicate aliquots of the plasmid-containing solution were collected and the relative abundance of the different plasmid conformations was analysed by gel electrophoresis. Plasmid pZR80(gfp), used in this study, was constructed by the insertion of a gfp-cassette coding for a Green fluorescent protein in the plasmid pZR80-2 [28]. Briefly, the 1.1-kb gfp-cassette was amplified by standard PCR by using pPnptII::gfp plasmid as template [29] and primers PnptII1F-SphI (5'-ATTATTGCATGCAACCGGAATTGCCAGCT-3') and TendR-SphI (5'-ATTATTGCATGCCCAATTCCTGGCAGTTTATG-3'), both containing a SphI restriction site (underlined) with 5' overhang. The SphI digested PCR product was ligated in the SphI linearized pZR80-2 plasmid and used to transform competent E. coli cells.Plasmid pZR80(gfp) was extracted from an overnight culture of the strain E. coli (pZR80(gfp)) with the QuiaPrep Mini Kit (Quiagen, Milan, Italy), quantified determining the optical density at 260 nm wavelength in a spectrophotometer (Beckman DU640). 212 μl of the plasmid preparation was inoculated to 10 ml of anoxic sterile filtered brines (final concentration 2 μg ml-1), and incubated in the dark at 15°C. At defined time intervals (figure 3) up to 32 days, a 100 μl aliquot of the plasmid containing brines were collected. All the inoculation and collection operations were conducted in an anoxic glove box. The recovered plasmid was desalted by dialysis against sterile milliQ water on a floating 0.1 μm pore size filter (25 mm diameter, Millipore, Milan, Italy) and stored at -20°C. At the end of the time course experiment 10 μl of each desalted aliquots were visualized by agarose gel electrophoresis in 0.5× TBE buffer stained with ethidium bromide. The relative abundance of the different plasmid conformations were estimated from the brightness of the plasmid bands in the electrophoresis gel picture using the software QuantityOne (Bio-Rad, Milan, Italy).Transformability of DNA after incubation into the brinesDesalted plasmid preparations recovered after incubation in the brines were used as donor DNA for the transformation of naturally competent cells of Acinetobacter baylii strain BD413, as described by Rizzi et al. [30]. Briefly, bacterial cells of A. baylii BD413 were recovered by centrifugation of 50 ml of an overnight culture in LB medium (Amersham Biotech, Milano, Italy), washed in sterile saline solution (NaCl 9 g l-1), resuspended in 10 ml of saline solution containing 15% (V/V) glycerol, divided in 100 μl aliquots and stored at -80°C. Cell concentration in each aliquot was 3.4 ± 0.8 × 108 cfu/ml. Sixteen μl of donor plasmid DNA were mixed with a thawed aliquot of naturally competent A. baylii cells, placed on a sterile mixed cellulose esters 0.22 μm pore size filter (47 mm diameter, Millipore, Milan, Italy), and positioned on the surface of an LB agar plate. After overnight incubation, the cells were recovered from the filter by resuspension in 5 ml of saline solution, serially diluted and plated on Luria Bertani (LB) (Amersham Biotech, Milan Italy) agar to count the total number of cells present on the filter, and LB agar supplemented with 100 μg/ml kanamycin to selectively count the transformant cells that acquired the plasmid. Transformation frequency was calculated as the number of KmR transformants over the total number of cells. Results are an average of triplicate experiments. Randomly selected colonies were checked for the expression of the gfp gene contained in the pZR80(gfp) plasmid, coding for a Green fluorescent protein, by epifluorescence microscopy (Zeiss Axioplan).Live/dead stainingCells from 200 μl of overnight cultures in medium PCB5% were collected by centrifugation (10 minutes 10000 g) and resuspended in 50 μl of sterile NaCl solution (5%). Five hundred μl of brine was added to the suspension. After 10 minutes of incubation 10 μl of the suspension was diluted with 100 μl of sterile water, and added with 3 μl of SybrGreen and 3 μl of propidium iodide solutions following the manufacturers instructions (Live/Dead staining kit, Molecular Probes). 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HallsworthJEYakimovMMGolyshinPNGillionJLMD'AuriaGde Lima AlvesFLa ConoVGenoveseMMcKewBAHayesSLHarrisGGiulianoLTimmisKNMcGenityTJLimits of life in MgCl2-containing environments: chaotropicity defines the windowEnviron Microbiol2007980181310.1111/j.1462-2920.2006.01212.x17298378\n23. DeFlaunMFPaulJHDetection of exogenous gene sequences in dissolved DNA from aquatic environmentsMicrob. Ecol198918212810.1007/BF0201169324196018\n24. MatsuiKHonjoMKawabataZEstimation of the fate of dissolved DNA in thermally stratified lake water from the stability of exogenous plasmid DNAAquat Microb Ecol200125147171\n25. SassAMMcKewBASassHFichtelJTimmisKNMcGenityTJDiversity of Bacillus-like organisms isolated from deep-sea hypersaline anoxic sedimentsSal Syst20084810.1186/1746-1448-4-8\n26. PolymenakouPNStephanouEGTselepidesABertilssonSOrganic matter preservation and microbial community accumulations in deep-hypersaline anoxic basinsGeomicrobiol J200724192910.1080/01490450601134283\n27. DemanécheSMonzorierLJChapelJPSimonetPInfluence of plasmid conformation and inserted sequence homology on natural transformation of Acinetobacter spAnn Microbiol2002526169\n28. RizziABrusettiLArioliSNielsenKMTamburiniASorliniCDaffonchioDDetection of feed-derived maize DNA in goat milk and evaluation of the potential of horizontal transfer to bacteriaEur Food Res Technol2008 in press\n29. StinerLHalversonLJDevelopment and characterization of a green fluorescent protein-based bacterial biosensor for bioavailable toluene and related compoundsAppl Environ Microbiol2002681962197110.1128/AEM.68.4.1962-1971.200211916719\n30. RizziAPontiroliABrusettiLBorinSSorliniCAbruzzeseASacchiGAVogelTMSimonetPBazzicalupoMNielsenKMMonierJ-MDaffonchioDStrategy for in situ detection of natural transformation-based horizontal gene transfer eventsAppl Environ Microbiol2008741250125410.1128/AEM.02185-0718165369"
+}

batch_11/PMC2531169.json

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+{
+    "id": "PMC2531169",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2531169\nAUTHORS: Anders Eriksson, Carl-Fredrik Flach, Anders Lindgren, Eva Kvifors, Stefan Lange\n\nABSTRACT:\nBackgroundThe cause and pathophysiology of ulcerative colitis are both mainly unknown. We have previously used whole-genome microarray technique on biopsies obtained from patients with ulcerative colitis to identifiy 5 changed mucosal transcripts. The aim of this study was to compare mucosal expressions of these five transcripts in ulcerative colitis patients vs. controls, along with the transcript expression in relation to the clinical ulcerative colitis status.MethodsColonic mucosal specimens from rectum and caecum were taken at ambulatory colonoscopy from ulcerative colitis patients (n = 49) with defined inflammatory activity and disease extension, and from controls (n = 67) without inflammatory bowel disease. The five mucosal transcripts aldolase B, elafin, MST-1, simNIPhom and SLC6A14 were analyzed using quantitative real-time PCR.ResultsSignificant transcript differences in the rectal mucosa for all five transcripts were demonstrated in ulcerative colitis patients compared to controls. The grade of transcript expression was related to the clinical disease activity.ConclusionThe five gene transcripts were changed in patients with ulcerative colitis, and were related to the disease activity. The known biological function of some of the transcripts may contribute to the inflammatory features and indicate a possible role of microbes in ulcerative colitis. The findings may also contribute to our pathophysiological understanding of ulcerative colitis.\n\nBODY:\nBackgroundUlcerative colitis (UC) is a disorder characterized by chronic mucosal inflammation of the large intestine. It is frequently associated with various extraintestinal manifestations. The inflammation may be limited to the rectum (proctitis), but mucosal lesions often continue more proximally (left-sided UC) or additionally embrace the transverse colon (extensive colitis) or the entire large bowel (pancolitis). The immune and cellular (non-immune) response is dysregulated in both the acute and the chronic phase of UC [1,2]. In Scandinavia, UC has been found to affect individuals of all ages, with an annual incidence of about 15 per 100 000 [3,4] and a prevalence of about 300 per 100 000 inhabitants [5].The pathogenesis and pathophysiology of UC are still under investigation [6]. We can tentatively say that the cause and onset of the disease is polygenic with environmental interaction; that is, there is a genetic predisposition [7-9] in combination with eliciting environmental factors which may precipitate the phenotype of UC [10]. In addition, interaction between the colonic epithelium and microbiological flora as well as a disintegrated mucosal barrier function may be important factors in the onset and development of UC [6]. The use of microarray technique analyses on mucosal specimens obtained from both patients with established UC and controls has allowed identification of candidate genes, which are valuable in research on UC pathogenesis. However, these UC candidate genes must be carefully selected, since recent evaluations of microarray data have revealed considerable divergence after examination of similar tissues [11-13]. Such divergent results are commonly presented in studies using pooled patient samples. In the present study however, the transcripts selected are based on our earlier individual whole-genome microarray screening and quantitative real-time PCR (RT-PCR) in patients with UC [14], where five changed genes/transcripts were identified; aldolase B, elafin, MST-1, simNIPhom (similar to NIP homolog), and SLC6A14. The pathophysiological properties of SimNIPhom have not yet been clarified, but the other transcript products have potential importance in secretion [15,16], anti-microbiological activity [17], and cell-mediated immune response [18].The primary aim of the present study was to define differences in the mucosal expression of five selected transcripts, retrieved from two different colonic locations in UC, by using a quantitative RT-PCR technique. We also aimed to evaluate the influence of ongoing anti-inflammatory treatment as well as the importance of the colonic UC extension and the severity class.MethodsPatients and tissue specimensBefore the colonoscopy procedure, consecutive male and female subjects (UC patients and controls, >18 y) were recruited to the present study. The UC diagnosis was based on the medical history, endoscopic findings, histological examination, laboratory tests, and the clinical disease presentation. The extent of UC and the clinical activity were classified in accordance with the Montreal Classification [19]. In brief, the colonic inflammatory involvement is defined as extension (letter E) combined with a number between 1–3 (E1 denotes proctitis, E2 left-sided UC, and E3 extensive colitis). In addition, the clinical severity grade (letter S) is defined. The S-score ranges from clinical remission (S0) to severe UC (S3). Mucosal biopsies were obtained from the rectum (10–15 cm proximal from anal verge) and caecum from all participants.No uses of corticosteroids, aminosalicylates, or immunosuppressants were registered in the control group, while 9 patients (18%) in the UC group were treated systemically with corticosteroids (prednisolone 10–20 mg). Twenty-seven UC patients (55%) were treated systemically with aminosalicylates (mesalazine 1.600–2.400 mg/24 h). Among these, two patients had additional ongoing therapy with aminosalicylate (mesalazine 500 mg QD) enemas and three patients were treated with corticosteroid enemas (prednisolone 37.5 mg QD or BID). Seven patients had stable (>3 months) ongoing immunosuppressant treatment (Azathioprine, 1.8–2.2 mg/kg bw).The remaining demographic and clinical data are presented in Figure 1.Figure 1Demographic and clinical data from the control and UC group respectively.RNA isolationThe biopsy specimens were immediately stored in RNA-later solution for isolation of RNA. The RNA-later-preserved biopsies were homogenized in a lysis buffer from the GenElute Mammalian Total RNA kit (Sigma, St. Louis, MO.) and total RNA was isolated according to the manufacturer's instructions. The RNA concentration was measured spectrophotometrically.Quantification by real-time polymerase chain reaction (RT-PCR)Two μg of total RNA from each sample were converted into cDNA. The cDNA synthesis was performed as described previously [20]. Oligonucleotide primers purchased from MWG-BIOTECH AG (Ebersberg, Germany) were used for the relative quantification (ABI-7500 system, software version 1.3) (Table 1). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene in all experiments. The expression level in each sample was compared with a calibrator by using the ΔΔCT- formula (ΔCT(calibrator) - ΔCT(sample)).Table 1Primers used for real-time polymerase chain reaction.GeneForward primerReverse primerAldolase B5'-aaggctgcaaacaaggaggcaacc-3'5'-tgaagagcgactgggtggaagcag-3'Elafin5'-tgtgaaggctcttgcgggatgg-3'5'-agggcagcagggacttaggaccag-3'SimNIPhom5'-cgccagacagctaggggagtgaag-3'5'-gcatttctgatattttgtgaccacgcac-3'SLC6A145'-gctgcttggttttgtttctccttggtc-3'5'-gcaattaaaatgccccatccagcac-3'MST-15'-aaccaggagtgtaacatcaagcaccgag-3'5'-cagttgtgggtaaagcaggcaagtgg-3'GAPDH5'-gagcaccaggtggtctcctctgacttc-3'5'-gccaaattcgttgtcataccaggaaatg-3'Statistical analysisDescriptive statistics and the Wilcoxon signed rank test (SAS, Statview®) were used. Median values are presented.EthicsThe study was approved by the local research ethical committee. All patients were given oral and written information before entering the study. Informed consent was obtained from all patients and controls.ResultsThe mean duration of UC was 9.3 years (proctitis 9.6 years, left-sided colitis 9.5 years and extensive colitis/pancolitis 9.2 years). Neither age nor gender was matched between the UC group and the control group.In order to evaluate any differences in transcript expressions within the control group (n = 67) with respect to background diagnoses (anaemia, diverticulosis, irritable bowel disease and polyposis), statistical analysis of each background diagnosis were compared to the remaining group of controls. No significant differences (p > 0.05) were detected for any of these diagnoses.Significantly higher transcript expressions of aldolase B and SimNIPhom and significantly lower transcript expressions of elafin, MST-1, and SLC6A14 were found in caecal biopsies in comparison to rectal biopsies from the control group (Figure 2). The only significant differences between rectal and caecal transcript expressions in UC patients were the decreased transcript expressions of elafin and SLC6A14 in caecal biopsies in comparison to rectal biopsies.Figure 2RT-PCR result (ΔΔCt(=ΔCttarget-ΔCtcalibrator)) for controls (filled dots) and UC patients (▲) presented as median values and 25th and 75th percentil (bars). * = p < 0.05, ** = p < 0.01, *** = p < 0.001, n.s. = non significant. Reference gene: GAPDH.Comparison of rectal biopsies from controls (n = 67) with rectal biopsies from UC patients with inflammatory activity in accordance with Montreal classifications S1–S3 (n = 28) showed significant elevations (p < 0.05) in UC patients of all transcript expressions with the exception of MST-1, which showed significantly (p < 0.05) decreased expression in UC patients. The same analysis of caecal biopsies from controls and patients with S1–S3 UC (n = 16) showed significantly elevated transcript expressions of aldolase B and SLC6A14 only. Distal biopsies from controls, compared with UC patients without inflammatory activity (S0), showed increased transcript expression in aldolase B only (median -1.62 vs. 1.0, p = 0,012). All other transcript analyses from both locations showed no significant differences (p > 0.05).All transcript analysis with respect to UC extension showed that left-sided (E2) and total colitis (E3) differed significantly from controls (p < 0.05); this was not the case for proctitis (E1).Statistical analysis concerning the influence of anti-inflammatory treatment on the transcript expressions within the UC cohort showed no statistical differences (p > 0.05) when comparing UC patients with ongoing corticosteroids (n = 9) or azathioprine (n = 7) respectively with the remaining UC patients. However, the 27 patients treated with mesalazine show a significant increase in aldolase B (median 0.48 vs. 3.02, p = 0.035) in comparison to the remaining UC patients.DiscussionGenetic predisposition, psychological stress, nutritional and environmental influences, intestinal pathogens and disturbed intestinal barrier function have all been proposeas pathogenetic factors in UC [6]. However, current knowledge about the pathogenesis and pathophysiology of UC [21] is incomplete. Moreover, with the exception of a few general serological inflammatory activity biomarkers, even less information is available regarding mucosa-associated transcript changes and their potential pathogenetic and pathophysiological role in UC [22]. This lack of knowledge may sometimes lead to uncertainty in diagnosis, judgement of prognosis and clinical management of UC patients.On the basis of exsisting knowledge of the biological functions of the transcripts investigated in this study, it is reasonable to believe that the demonstrated alterations might be related to predisposition and/or the pathophysiological response in UC.It is intriguing that aldolase B and SLC6A14 were up-regulated in rectal as well as caecal mucosa in UC compared to controls. Aldolase B is known to be mainly expressed in the intestinal villus cells and it has a central role in the glycolytic pathway. It also participates in regulation of intestinal secretion [16]. Since SLC6A14 is also known to encode a Na+/Cl- driven amino acid transporter B(0+) [15], the up-regulation of aldolase B and SLC6A14 might be a common pathophysiological response, aimed at counteracting the exaggerated loss of fluid seen in UC. Theoretically, the up-regulation of these two transcripts could be a local response to the increased feacal/fluid stream, where bioactive molecules comprise ability to regulate transcript expression. Additionally, since the inflammatory activity and load of fluid over time is usually most pronounced in the distal part of the colon, the registered changes in aldolase B and SLC6A14 may reflect long-term inflammatory activity. Our finding that aldolase B from distal biopsies is significantly elevated during the remission phase (S0) indicates that the regulation of this transcript not only is secondary to the inflammatory activity.The involvement of the microflora and its importance in the onset, development and preservation of UC has been discussed [6]. The SLC6A14 transcript expression is therefore also interesting in this respect, since it is involved in the host's antibacterial response [21]. In addition, the defensine-like epithelium associated antimicrobial molecule elafin, antagonizing human neutrophil elastase preventing tissue injury via inhibition of excessive release of proteolytic enzymes from inflammatory cells is interesting in this context [18]. The present results confirm an elafin transcript enhancement in caecal as well as rectal biopsies from patients with UC. Thus, the combined elevation of elafin and SLC6A14 may contribute to an amplified defence reaction aimed to restoration and maintenance of the mucosal integrity. This finding may indicate a pathogenetic role of the microflora in UC.MST-1 was included in the present study due to its alterations in UC, as shown in our previous experiment [14], although it was excluded from that publication due to deviation in its control group. MST-1 is known to be capable of inhibiting cell-mediated immune responses via down-regulation of IL-12 production and subsequently inhibition of macrophage activation [23]. Consequently, the observed down-regulation of MST-1 in rectal specimens may contribute to an enhanced cellular immune response in UC. A reasonable explanation of the concomitant decreased MST-1 transcript expression and increased aldolase B, SLC6A14, and elafin transcript expression is that the changes describe a pathophysiological response to a more pronounced inflammatory and, possibly, an exaggerated microbial load in at least the rectal part of the colon mucosa.The fifth identified significantly up-regulated transcript (in rectum only) SimNIPhom (similar to the numb-interacting homolog), encodes a hypothetical protein, at present of unknown pathophysiological importance.Our results supports that specimens from the rectal mucosa are more suitable for further analysis of the selected transcripts, due to the more predictable inflammatory involvement in the rectum and its availability for direct inspection and easy biopsy sampling.Our data can not answer whether the observed changes in expressions of the five selected transcripts may be present in e.g. other inflammatory, infectious or autoimmune conditions since this study uniquely focused on UC patients compared to non-inflamed controls.ConclusionThe five changed gene transcript expressions have relation to UC, its extension and clinical severity. Whether the presented results will contain discriminative potential of importance for the medical care of patients with UC in future clinical practice remains to be elucidated.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsAE designed the study, preformed sampling of biopsies, analyzed the data, and prepared the manuscript. C-FF analyzed the data. AL preformed sampling of biopsies. EK coordinated the study. SL designed the study, analyzed the data and prepared the manuscript. All authors read and approved final manuscript.Pre-publication historyThe pre-publication history for this paper can be accessed here:\n\nREFERENCES:\nNo References"
+}

batch_11/PMC2531179.json

@@ -0,0 +1,4 @@
+{
+    "id": "PMC2531179",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2531179\nAUTHORS: Jenny Andersson, Pär Salander, Marie Brandstetter-Hiltunen, Emma Knutsson, Katarina Hamberg\n\nABSTRACT:\nBackgroundIn many diseases men and women, for no apparent medical reason, are not offered the same investigations and treatment in health care. This may be due to staff's stereotypical preconceptions about men and women, i.e., gender bias. In the clinical situation it is difficult to know whether gender differences in management reflect physicians' gender bias or male and female patients' different needs or different ways of expressing their needs. To shed some light on these possibilities this study investigated to what extent it was possible to identify patients' sex when reading their blinded illness narratives, i.e., do male and female patients express themselves differently enough to be recognised as men and women without being categorised on beforehand?MethodsEighty-one authentic letters about being diseased by cancer were blinded regarding sex and read by 130 students of medicine and psychology. For each letter the participants were asked to give the author's sex and to explain their choice. The success rates were analysed statistically. To illuminate the participants' reasoning the explanations of four letters were analysed qualitatively.ResultsThe patient's sex was correctly identified in 62% of the cases, with significantly higher rates in male narratives. There were no differences between male and female participants. In the qualitative analysis the choice of a male writer was explained by: a short letter; formal language; a focus on facts and a lack of emotions. In contrast the reasons for the choice of a woman were: a long letter; vivid language; mention of emotions and interpersonal relationships. Furthermore, the same expressions were interpreted differently depending on whether the participant believed the writer to be male or female.ConclusionIt was possible to detect gender differences in the blinded illness narratives. The students' explanations for their choice of sex agreed with common gender stereotypes implying that such stereotypes correspond, at least on a group level, to differences in male and female patients' illness descriptions. However, it was also obvious that preconceptions about gender obstructed and biased the interpretations, a finding with implications for the understanding of gender bias in clinical practice.\n\nBODY:\nIntroductionGender plays an important, but not necessarily appropriate part, in medical decision-making. Research has shown that differences between men and women regarding biological processes, socioeconomic conditions, risk behaviour and environmental risk factors may all contribute to differences in health [1,2]. Thus it is sometimes appropriate to investigate and treat male and female patients differently. On the other hand, there is also evidence that women, for no apparent medical reasons, are not offered the same treatment as men, which raises the possibility of gender bias. For example, many studies show that women are less likely than men to receive more advanced diagnostic and therapeutic interventions [3-7]. In the clinical situation it is often difficult to know the extent to which gender differences in management reflect physicians' gender bias or are due to other physician, patient or communication characteristics related to gender [8-10].Patients' wishes and communication behaviour contribute to gender differences in health care [11,12]. It is argued, for instance, that men describe their symptoms in a straightforward and demanding way while women often act in a submissive way during consultations, give vague descriptions of their symptoms and are hesitant to accept potentially dangerous measures such as surgery [7,13]. In studies of psychosocial adaptation to cancer a recurring result is that men, more than women, prefer to share information while women tend to adapt to the stressful situation by expressing and sharing emotions [14,15]. However, even if there is some tendency for men to be information-oriented and women emotion-oriented, the research results are more complex. There are reports showing that men experience distress when there are social constraints to express emotions [15], that the need for information is more pronounced among women [16], and that women are more knowledgeable about their disease than men [17].Perceptions about gender differencesAssumptions and beliefs about differences in men and women regarding behaviour, skills, emotions and needs are widespread in society. Assumed gender differences are often polarized as opposites, for example: males are associated with order, control and individualism while females are considered incapable of controlling their feelings and as having a natural sense for the family [18,19]. The man is seen as strong and active, and the woman as weak and passive; the man is symbolised by work, while the woman is associated with the home. In research, generalisations and stereotypes about men and women and other social groups are mainly treated as problematic since they bias interpretations of human activities and are sources of discrimination [20]. However, there is also an ongoing discussion about how much truth there might be in a given stereotype [21].It is debatable whether there is a male and a female \"language\", but research shows that there are often gender differences in the way language is used [22,23]. Analyses of conversations show fairly clearly that men and women talk differently. Issues such as turn-taking, politeness, interruption, use of swear words etc. are distinctly gender marked and may be read as signifying power relationships [23-25]. Studies also provide evidence of differences in the way men and women write [26,27]. In her research on autobiographies, Mary Gergen found substantial differences in the narratives written by men and women. Men focused on the career and achievements of the subject [26]. Emotional ties were mentioned only as 'facts', i.e., male authors did not try to recreate in the reader emphatic emotional responses. On the other hand, in narratives written by women the career line was important but was mingled with other issues that had great personal impact. Furthermore, women's autobiographies dealt extensively with relationships with others.Discourse processes have developed out of what and how things are told, who speaks (characterised for example by gender, age, ethnicity, education and social position), and who listens (characterized according to the same factors). Similarly, a gender perspective on written language includes not only the author and the narrative itself but also the reader's interpretation. Since readers of a text are usually aware of whether the author is a man or woman, their expectations and interpretations might be affected by their preconceptions about gender [26]. In an experiment involving an authentic text in two versions, identical in all but the simulated male or female author, differences were shown in how readers viewed the author. Based on the same text, the male writer was considered more trustworthy and intelligent and the female writer more humane [28]. This shows that preconceptions have a great influence on how a text is judged and gendered expectations of manstories and womanstories might be more important than content and facts [26].In a previous study we investigated gender differences in 83 patients' letters concerning their experiences when being diagnosed with cancer [29]. It was found that more women than men wrote long, personal and emotional narratives, thus confirming the earlier results about gender differences in communication behaviour described above. However, the majority of letters, about 60%, were neither long, personal or emotional, nor short, impersonal or unemotional, and thus they were hard to categorize. When discussing these results we asked if the gender differences were significant enough to be detectable if all obvious hints about the patient's sex were removed (i.e., pronouns and expressions like \"my wife\" and \"my husband\")? If so, this would confirm that there are genuine differences in male and female patients' descriptions, differences that are not just creations of the reader knowing the sex of the patient-writer on beforehand.The aim of the present paper was, therefore, to investigate the extent to which it was possible to identify the patients' sex by reading the same letters, once all information regarding the sex of the patient-writer had been removed. Students of psychology and medicine were invited to be participants. In order to provide nuances to the results, and hints about how gender was created, the students' explanations of their choice of sex were scrutinized in a few of the letters where the students had varying rates of success in determining the authors' sex.MethodLetters from patientsThe study was based on letters written by patients with a recent diagnosis of cancer. The letters were collected at an oncology department in Sweden during a five-month period in the late 90s, in order to analyse the manner in which the patients had received their diagnosis. All patients aged 18–70 who had received their cancer diagnosis 2–8 months previously, were asked to \"...write a page or two describing how you received your diagnosis... including what the physician told you, how you reacted and how you felt afterwards. In addition, please describe both what you perceived as beneficial and what was detrimental...\" Out of 187 consecutive patients invited, 138 (74%) submitted a written narrative [30].In the present study, all the letters about breast cancer (n = 53) were excluded since this group of patients are subjected to a mammography-screening programme that was considered difficult to blind. Two letters were removed because they were illegible. The remaining letters (n = 83) were typed and all names, places and dates were systematically changed to prevent identification of patients, doctors and others concerned. To blind the letters all information that revealed the patient's sex was removed. The words \"husband\" and \"wife\" were consistently changed to \"co-habiter\", \"mother\" and \"father\" were changed to \"parent\". Detailed descriptions with references to specific clinics or surgical procedures were made less specific if they provided clues to the patient's sex. For example, any references to prostate or gynaecological symptoms or statements about women's clinics or urology departments were changed or removed. Abbreviations and spelling mistakes were retained as in the original letter. During this process two letters were excluded since it was considered too difficult to change them without distortion. Eighty-one letters remained, 42 written by men and 39 by women. The ethics committee of Umeå University approved the study.ParticipantsA total of 130 participants, 87 medical students and 43 psychology students at Umeå University, volunteered to take part in the study that was carried out on five occasions during the fall of 2005. The participants were aged 18 to 42 years (M = 24.4); 45 of them were men and 85 women.In a pilot study we found that it was too demanding and time consuming for participants to read 81 letters. Therefore, two test-groups of participants where formed, groups A and B, each reading one half of the letters. The goal was to achieve an equal distribution of participants with respect to the number and sex in the two test groups (Table 1).Table 1Distribution of letters and characteristics of participants in the test groups.Letters (N = 81)Participants (N = 130)NumberWritten by men/womenNumberMen/WomenMedicine/PsychologyGroup A4120/216322/4143/20Group B4022/186723/4444/23Data collectionThe participants were informed that the study was based on patients' authentic stories. Each participant was first asked to answer questions about their own sex, age and social background. They then read the letters belonging to their test group. For each letter they were asked to make a decision about the patient's sex (man or woman) and to explain their choice of sex in an open-ended question. The participants were instructed to read through the letters rapidly and make decisions based on their first impressions.The current paper focuses on the decision about the author's sex and uses the explanations for the choice of sex in four letters to illuminate the complexity in the findings.Missing dataIn total there were 5263 decisions about sex to be made, but data were missing for 79 of these decisions spread over letters and male and female participants. There is no reason to believe that this gap had any systematic influence on the results.AnalysisThe relationships between participant characteristics (sex and discipline [medicine or psychology]) and participant success rate for the sex-decision task were analysed using the statistical program SPSS 11.0 for Windows. Unpaired t-tests and ANOVAs where used to compare differences between means. The level of significance was set at p < 0.05.In order to shed more light on the statistical findings four letters were analyzed further. The letters that were selected were those with the highest and lowest frequencies of correct decisions about sex, together with two letters where about 50% of the participants' hade made a correct decision (see* in Table 3). For these letters the open-ended answers about the motives for the decision about sex were read and coded by three of the researchers (JA, MB-H, EK). In a joint session with all five researchers, the codes were compared, discussed and sorted into the broad categories: length, language and content. In a few cases of disagreement about how to categorize, the researchers discussed to find a solution. In this paper the motives for choosing a male or female patient are presented in typical examples, to illustrate the reasoning connected with each letter.ResultsQuantitative analysisAn independent t-test showed that there were no significant differences between the results of the medical and psychology students (p = 0.377) nor between the average numbers of correct decisions about sex in the test groups A and B (p = 0.250). There were also no significant differences between the success rates of male and female participants (p = 0.628).Table 2 shows the percentage of correct decisions for all letters and male and female letters, made by all participants, and by male and female participants separately. The mean value for correct decisions among all participants for all letters was 61.7% with a variation from 26.8% to 82.5%. Comparing these results to chance, i.e. 50% correct decisions, independent t-tests showed that the participants were significantly better than chance in their judgements of male as well as female letters (p-values not shown in the table). The participants also chose 'male patient' significantly more often than 'female patient' (p < 0.000).Table 2Percentage of correct decisions about patient's sex made by male and female participants.All letters (N = 81)Male letters (n = 42)Female letters (n = 39)p-values*Male participants (n = 45)61.265.755.9< 0.000Female participants (n = 85)62.064.858.5< 0.000All participants (N = 130)61.765.258.0< 0.000* p-values comparing the success for male and female letters.Table 3 shows the distribution of letters according to the proportions of correct decisions made. For six of the 81 letters (7.4%) the proportion of correct decisions was lower than 30%, for 48 letters (59.3%) it was between 30 and 70 %, and for the remaining 27 letters (33.3%) the proportion of correct decisions was higher than 70%. Both male and female participants had a higher success rate on male than on female letters (p < 0.000).Table 3The letters sorted according to the percentage of correct decisions made about the patient's sex.Correct decision interval (%)Letter labelNumber of letters (%)Patient's sex†0 – 5.05.1 – 10.032*1 (1.2)210.1 – 15.0591 (1.2)215.1 – 20.020.1 – 25.025.1 – 30.04, 18, 25, 664 (4.9)2, 2, 2, 230.1 – 35.044, 79, 36, 37,4 (4.9)1, 1, 1, 235.1 – 40.0681 (1.2)140.1 – 45.058, 55, 573 (3.7)2, 1, 145.1 – 50.033, 81, 6, 23, 75, 2, 22, 74*8 (9.9)2, 2, 1, 1, 2, 1, 2, 150.1 – 55.045*, 50, 53, 54, 30, 396 (7.4)2, 1, 2, 1, 2, 155.1 – 60.034, 13, 193 (3.7)1, 2, 260.1 – 65.015, 29, 38, 48, 63, 5, 20, 46, 43, 62, 65, 73, 8013 (16.0)2, 2, 1, 2, 2, 2, 1, 2, 1, 1, 2, 2, 165.1 – 70.010, 14, 72, 56, 60, 69, 1, 11, 17, 3510 (12.3)2, 2, 1, 1, 1, 2, 1, 2, 1, 170.1 – 75.076, 7, 21, 52, 77, 316 (7.4)1, 1, 1, 2, 1, 175.1 – 80.064, 78, 3, 9, 26, 41, 517 (8.6)2, 1, 2, 1, 1, 2, 180.1 – 85.061, 71, 8, 40, 495 (6.2)2, 2, 2, 1, 285.1 – 90.027, 12, 47, 67, 28, 42, 707 (8.6)1, 1, 1, 1, 2, 1, 190.1 – 95.0161 (1.2)195.1 – 10024*1 (1.2)2Total81 (100)†1 = man, 2 = woman*Letters further examined in the qualitative analysis.All six letters with a success rate below 30% were written by women. A majority of the letters (17/27) with a success rate above 70% were written by men.Qualitative examinationThe students' explanations as to why they believed the author was a male or female patient varied from just a few words, e.g. \"short letter\" or \"lot of emotions\", to three or four sentences where they expressed several reasons and reflections. In the following, the letter on which the participants had the lowest proportion of correct decisions about sex is labelled 'the most difficult letter', the one with the highest proportion of correct answers is 'the easiest letter', and the two letters where the participants were quite divided on whether the author was a male or female patient are labelled the 'in-between letters'.The most difficult letterLetter 32, written by a woman, was very short and consisted of only two sentences:\"My cancer was detected in the following way: I had a severe cough and cold and blood started to come from the rectum.\"Sixty participants read this letter. Five made a correct decision about sex and two of them explained their choice. They referred to \"the sentence structure\" and the disclosure of medical facts strongly linked to personal integrity as reasons for believing it was written by a woman patient.Fifty-five participants thought that a man had written the letter and 46 of them explained their choice. Their reasons for a male author could be summarized as follows: The narrative was short and contained factual information and no emotions; the patient seemed dissociated from feelings and unwilling to share experiences and thoughts; several participants claimed that a female writer would have given a more balanced description and put some more effort into the assignment.The easiest letterThe letter where the sex of the author was correctly identified most often (number 24), was written by a woman. It was longer than most letters, at 408 words, and was written in a format that described events in temporal order as well as the patient's reactions to these events. The patient mentioned family and friends, described medical staff and openly shared personal thoughts and feelings.All 61 participants who read this letter correctly judged that the patient was a woman and 58 explained their choice. The length of the letter was often mentioned, the language was described as soft and vivid and the use of descriptions such as \"smooth and gentle\" about the male oncologist were frequently given as the motive for choosing a female writer. That the patient shared emotions, mentioned weakness and tears, described family members, friends and medical staff, and emphasized the meaning of support and network, were all seen as clues to the sex of the author. The attitudes of those around the patient were also seen as indicating a female author, e.g. when the patient was informed about the diagnosis, the doctor embraced and held the patient's hands.The two 'in-between' lettersLetter 45 was written by a woman. The letter was 376 words long and started with the following sentence: \"You have asked for my experiences concerning the manner in which I received the information that I had malignant cell changes\".Following this the patient gave a detailed description of the course of events and the feelings involved. The letter also contained observations concerning positive and negative experiences of her treatment and how she was met by the staff.Sixty-seven participants read this letter, and 35 of them (52%) correctly decided it was written by a woman. Sixty explained their choice. Participants who believed the author was a woman found the letter long and detailed. The language was described as proper, with an introduction ensuring that the reader did not forget why the letter was written. Some participants referred to the patient's use of the Swedish word \"gräsligt\", (\"horrible\" in English), as hinting that this was a female patient. The narrative was seen as emotional with disclosures of feelings of weakness and fear, seen as strong hints of a 'female writer'. Other reasons related to content concerned \"the extensive description of the course of events\" and the way the patient reflected on and analyzed the course of events.Participants who thought that the writer was a man found the introduction formal. The sentences were described as short and the language academic. The use of the Swedish word \"pallade\" (\"managed\" in English) was associated with a man. The narrative was described as carefully prepared, based on factual information and focused on events rather than emotions. It gave the participants the impression of an evaluation, written on order. The patient seemed energetic and put more trust in himself than in those around him.Letter 74 was written by a man. It consisted of 110 words and started with a description of the patient's shock when the black spot on the arm was diagnosed as a malignant tumour. The writer mentioned the doctor by title and full name and gave him credit for how the bad news was delivered. The patient described feelings of depression following the diagnosis. However, after receiving psychoactive drugs the patient felt a lot better and had returned to work.Thirty-tree of the 67 participants (49%) who read the letter thought that the writer was a man while the other half thought it was a woman. Fifty participants explained their decision. Those who thought it was a man described the narrative as short and concise, distant and formal. They stated that the patient focused on the disease as a diagnosis rather than on the emotions involved. Other explanations focused on the patient's comment that it was important to get back to work, and the long waiting time before seeing a doctor.The participants who believed it was written by a female patient thought, on the other hand, that a long waiting time to see a doctor indicated that the patient was a woman. They also found hints about gender in \"the style\" and \"the choice of words\". Other clues mentioned were how the patient referred to the doctor by title and full name and the comments about reception. But the most common motives for choosing 'female patient' were that the patient described anxiety and weakness and was not afraid to ask for help or medical treatment for depression.DiscussionSummaryThe results showed that in 62% of the cases the university student participants succeeded in identifying the sex of the patient who had written the letter, which was significantly more accurate than a chance allocation. Male and female students did not differ in this regard. The success rate varied between letters and for one third of the letters more than 70% of the participants made a correct decision. For the remaining two thirds of the letters many participants thus had problems identifying the patient's sex. There were significant differences between the students' success rate for male and female letters, with more correct decisions being made for male letters. In four letters the explanations for the choice of sex were analysed and the participants based their choices on three factors – length, language and content. The participants were more likely to say that a particular letter was written by a man if the letter was short, the language was more formal and academic, and the content focussed on the factual info. If the letter was long, written in more expressive manner, and described emotions and relationships the participants were more likely to decide that the author was a woman. However, depending on whether the participants believed the patient was a man or a woman, the same utterances and expressions were interpreted in different ways.On methodThat the narratives were authentic and written by \"real patients\" with cancer, as opposed to constructed paper cases, increased the credibility of the study. Further, the letters were not initially collected with a gender study in mind but to study the communication of bad news. This fact, presumably, limited the risk that the patients consciously adjusted their narratives according to societal norms about male and female patients' behaviour.In research comparing men and women there is always a risk of circular arguments. Men's and women's behaviours, thoughts or narratives are compared, and differences and similarities are noted. This raises the question of whether the interpretations are true differences or biased by the observers' preconceptions and expectations of a gendered pattern. A strength in this study was the use of the written narrative form, making it possible to create \"neutral\" patients in the sense that there was no obvious information within the text that revealed the patient's sex. On the other hand, differences between men and women in writing about illness might not be the same as differences when talking about illness, for example when seeing a physician. The interpretation of gender may be different in reading compared to listening. Thus the design with neutral patients and written narratives inherited weaknesses along with the strengths.The students' participation in the study was voluntary and more women than men took part. However, comparisons of the results showed no significant differences in the responses of men and women students. The gender topic of the study may have contributed to a preponderance of male and female students with a special interest in gender issues. Whether this influenced the results is beyond our knowledge, but even participants aware of gender issues had to rely on their preconceptions and beliefs when sorting the letters.The instructions to read through the letters rapidly and make judgements based on their first impressions forced the participants to be categorical. Many found this unpleasant, indicating an aversion towards using categorical generalisations. In studies of stereotypes and attitudes it is regularly found that people are inclined to express themselves in politically correct terms and try to distance themselves from gender stereotyping [31]. Yet, on an unconscious level they nevertheless rely on the stereotypes they are trying to avoid. Thus, if our students had felt less pressure it is likely that the same stereotypes would have emerged, but probably in more guarded terms.On resultsBetter than chance allocationThe participants made accurate decisions about the sex of the author in approximately 62% of the cases and in one third of the letters the success rate was even higher. These results indicate, hardly surprisingly, that there were gender differences in the illness narratives and confirmed our findings from a previous study that differences between male and female letters are detectable on a group level [29]. This finding is consistent with other studies that show fairly stable gender differences in conversations and use of language [23-26]. Reliable gender differences have also been found in meta-analyses of behaviours and traits in areas such as cognitive performance, cognitive attitudes, personality and group behaviour [21]. However, the fact that the students in our study were able to recognize gender differences, i.e. to make accurate decisions about the patient's sex in a majority of the blinded narratives, shows that knowledge and awareness of gender differences is common and widespread among people.It is hard to know whether he success rate would have been different had the participants been qualified psychologists or physicians with more clinical experience. In a study with a similar design, English professors in fact did worse than college students when they were asked to identify whether a man or a woman had written different 100-word passages of American fiction [27]. Experience in reading or analysing texts did not increase their ability to categorize the author by sex.In our study the success rate varied greatly across the letters showing that although preconceptions about gender differences rest on a basis of 'reality' on a group level, there are many exceptions and variations on the individual level. This fact illustrates the risk of making prejudiced assessments and biased interpretations in everyday communication.The lack of a sex difference in students' ability to identify the patients' sex, i.e. to recognize gendered patterns in the narratives, is in line with previous research showing that on the whole men and women are aware of gender differences in behaviour to the same extent [21] and have similar associations and preconceptions about gender [31,32].The participants were more successful at identifying which letters were written by male than female patients. One possible explanation for this might be related to 'the male norm', i.e., that men and behaviours associated with men are seen as the norm and the point of reference while women, and their needs and behaviour, are seen as exceptions in many situations [33]. One consequence of this might be that the participants were more inclined to see a man in the narratives and more prone to guess on a male writer when they were unsure. This would also explain why the participants chose 'male patient' more often. Another possible explanation is that women tend to show a greater capacity to vary their text than men, and to a greater extent adjust their language to existing circumstances [26]. In a social system where the man is the norm and women run the risk of being disregarded or not taken seriously, there might be a purpose behind women adjusting their language to a male language norm [24]. Some female writers may employ a discourse that seems like male writing, or at least seems to be in line with how readers are used to seeing male writing [27].The decision processWhen comparing the reasons for choosing a male or female writer in letters with the highest and lowest success rates, pros and cons of applying common generalizations about men and women in individual cases are illustrated. Letter 24 was written by a woman and fulfilled preconceptions about women's writing regarding length, personal and emotional content, and inclusion of family members and other people in the text. Subsequently, all participants succeeded in correctly identifying the author as a woman. Letter 32 was difficult since it was written by a woman whose letter did not fit into the stereotypes about women's way of communicating. On the contrary, she wrote 'like a man'. The participants commented that the author was 'writing briefly' and 'with factual information and no emotions', characteristics they associated with a male author. This indicates that the letters with very high rates of successful allocation corresponded with generalizations about gender while those with very low success rates clearly deviated from gender stereotypes.The reasons underlying the allocation of the 'in-between' letters suggest that the participants were sifting and weighting a variety of factors when categorizing the author by sex. It appears that they created an image of the narrator's sex from some information they initially gleaned, and then they looked for clues to confirm their belief about the writer's sex. We do not know, however, to what extent these initial clues concerned 'length', 'language', 'content', or even something else, and we do not know the hierarchy governing these clues, i.e., was a 'long narrative' more likely to supersede the use of 'academic language' or 'emotional expressions' as cueing the initial impression that formed the basis for the decision. These are interesting questions that remain to be considered in forthcoming research. It was, nevertheless, striking that the same phenomenon or expressions were interpreted in quite different ways depending on whether the participant decided that the patient was a man or a woman. For example: the content in letter 45 was described as \"emotional\" or \"reflective and analysing\" by the participants who thought the author was a woman. The same content, on the other hand, was commented on as \"based on facts and events\" or \"written on order like an evaluation\" by the participants who thought the writer was a man. In the process, interpretations of identical utterances were thus biased by the participants' gendered preconceptions. These findings correspond with the results from earlier experiments, where identical articles gave the readers quite different views of the author depending on whether the simulated author was man or woman [28].Our results were gathered in a study with an experimental design and the participants were students. It is reasonable to believe that similar interpretative processes take place in a clinical situation. In the clinic there are seldom doubts about a patient's sex and, depending on whether the patient is a man or a woman, the clinician has different preconceptions and expectations about the story that will be told. Thus, when a male patient describes his symptoms, needs and experiences, it is likely that the doctor, psychologist, or other health care staff member, will interpret and remember the narrative differently compared to when a female patient tells the same story [34,35]. This might be one clue to the mental processes causing the gender bias in diagnoses and treatment identified in many studies in various fields of health care [3-7,36,37].The participants' explanations for their decisions contained many stereotypes, preconceptions and ideas about men and women. In the black-and-white generalizations that the participants uses the categories man and woman stood out as complete opposites defined by their difference. For example, participants explained their choice by comments such as \"a woman wouldn't express herself this way\". However, if stereotypical assumptions and generalizations were completely false and there were no differences between male and female writers, the participants would not have done significantly better than chance allocations in their judgements. Generalizations about men and women are based on individual experiences as well as abstractions and conceptions in society, and stereotypical ideas often contain some truth when compared to results from observational studies and other research [21]. In fact, people use generalizations and preconceptions as an aid in understanding the world. Without generalisations we would be lost in a fragmented social world that was difficult to understand. However, generalizations and stereotypes are also problematic as they bias what we see and hear; they imply a risk of neglecting variation, less well-known aspects, and of making skewed assessments.To ensure that students in the health care field are aware of the risk of interpreting patients' behaviour in ways that reflect gender bias it seems essential to include reflections about the impact of preconceptions and generalisations in medical education. In healthcare, it is also vital to safeguard a working climate where generalisations and stereotypical attitudes towards men and women (and also other social groups, e.g. immigrants, unemployed, elderly and disabled) are constantly questioned and reflected on in clinical discussions among doctors, psychologists and other healthcare staff. The results from this article could contribute to such discussions, and the different interpretations of identical expressions depending on whether the author was taken for a man or a woman might be used as examples.More knowledge about the cognitive, behavioural and communication processes leading to gender bias in medical work is needed. Observations of authentic consultations in different clinics would be valuable [38]. In a future study, we will focus on the explanations for the participants' choice of sex in more of the 'in-between' letters.ConclusionIt was possible for participants to detect gender differences in patients' illness narratives although the narratives were blinded. The reasons the participants gave for their sex-categorisation reflected common gender stereotypes suggesting that such stereotypes are more than problematic clichés; at least on a group level they correspond to differences in male and female patients' illness descriptions. However, it was also obvious that preconceptions about gender obstructed and biased the participants' interpretations of the letters. Depending on whether the participants believed the author was a male or a female patient they interpreted the same utterances in different ways. Translated into the clinical situation our results suggest that on the one hand there are gender differences that are recognizable and useful in clinical work consistent with common gender stereotypes. On the other hand, stereotyped preconceptions and generalisations about gender imply that there is a risk that health care staff might interpret a story told by a male patient differently than the identical story told by a woman, and this could result in gender biased investigations and treatments. These findings are important for further understanding of and research about gender bias processes in clinical work.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsAll five authors contributed to the design, data collection and drafting of this article. JA, PS and KH were mainly responsible for the final drafting. All five authors read and approved the manuscript.\n\nREFERENCES:\nNo References"
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+{
+    "id": "PMC2531184",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2531184\nAUTHORS: Rupert CM Jones, Maria Dickson-Spillmann, Martin JC Mather, Dawn Marks, Bryanie S Shackell\n\nABSTRACT:\nBackgroundGuidelines on COPD diagnosis and management encourage primary care physicians to detect the disease at an early stage and to treat patients according to their condition and needs. Problems in guideline implementation include difficulties in diagnosis, using spirometry and the disputed role of reversibility testing. These lead to inaccurate diagnostic registers and inadequacy of administered treatments. This study represents an audit of COPD diagnosis and management in primary care practices in Devon.MethodsSix hundred and thirty two patients on COPD registers in primary care practices were seen by a visiting Respiratory Specialist Nurse. Diagnoses were made according to the NICE guidelines. Reversibility testing was carried out either routinely or based on clinical indication in two sub-samples. Dyspnoea was assessed. Data were entered into a novel IT-based software which computed guideline-based treatment recommendations. Current and recommended treatments were compared.ResultsFive hundred and eighty patients had spirometry. Diagnoses of COPD were confirmed in 422 patients (73%). Thirty nine patients were identified as asthma only, 94 had normal spirometry, 23 were restrictive and 2 had a cardiac disorder. Reversibility testing changed diagnosis of 11% of patients with airflow obstruction, and severity grading in 18%. Three quarters of patients with COPD had been offered practical help with smoking cessation. Short and long-acting anticholinergics and long acting beta-2 agonists had been under-prescribed; in 15–18% of patients they were indicated but not received. Inhaled steroids had been over-prescribed (recommended in 17%; taken by 60%), whereas only 4% of patients with a chronic productive cough were receiving mucolytics. Pulmonary rehabilitation was not available in some areas and was under-used in other areas.ConclusionDiagnostic registers of COPD in primary care contain mistakes leading to inaccurate prevalence estimates and inappropriate treatment decisions. Use of pre-bronchodilator readings for diagnosis overestimates the prevalence and severity in a significant minority, thus post bronchodilator readings should be used. Management of stable COPD does often not correspond to guidelines. The IT system used in this study has the potential to improve diagnosis and management of COPD in primary care.\n\nBODY:\nBackgroundChronic obstructive pulmonary disease (COPD) is a growing burden to patients and the National Health Service. Disease progression is preventable by early detection combined with smoking cessation [1,2]. The annual costs are estimated at £1,639 per patient, 54% of this is for unscheduled care relating to exacerbations [3]. In primary care, earlier diagnosis and the use of interventions aimed at preventing exacerbations and delaying the progression of the disease may be the best way to tackle these costs [4].The guidelines of the National Institute for Health and Clinical Excellence (NICE) on COPD diagnosis and management [5] are clear and comprehensive. However, major problems regarding the practical implementation of these guidelines have been identified that may affect the accuracy of COPD diagnosis and treatment.Spirometry is a fundamental component of COPD diagnosis [5,6], and can be performed accurately in primary care [7]. However, some studies have found major problems with existing spirometry [8-11]. Up to one third of practices had no spirometer, in addition to which practice nurses lacked training and support in performing spirometry and interpreting the results [9].At present, neither the NICE nor the GOLD guidelines recommend reversibility testing in the diagnosis of COPD. Post-bronchodilator spirometry is necessary for diagnosis of COPD according to the GOLD guidelines [6], but not the NICE guidelines [5].Reversibility testing using bronchodilators affects the frequency of diagnoses of COPD [12,13]. Prevalence of COPD was 27% lower using post-bronchodilator spirometry compared to spirometry without bronchodilator [12].Reversibility testing may also influence treatment decisions. For example, based on a study that used post-bronchodilator readings [14], inhaled steroids are recommended for patients who have a forced expiratory volume in one second (FEV1) ≤ 50% predicted and two or more exacerbations per year [5]. Thus, failure to use bronchodilators may cause some patients to be given treatment they do not need.There is widespread evidence of poor adherence to guidelines in the management of COPD [15-20]. Studies in primary care have demonstrated problems with provision of most treatments including medications, vaccination, smoking cessation advice and referral to pulmonary rehabilitation. For example, 25% of general practitioners (GPs) systematically prescribed inhaled corticosteroids to patients with severe COPD, but nearly half of them were unaware of the guideline-based indication for steroids [19]. In the United Kingdom, pulmonary rehabilitation is available to only 2% of those who need it [21]. Provision of advice on smoking cessation is also poor. Hyland et al. [22] reported that more than one third of current smokers with COPD had not been offered help with smoking cessation such as referral to a smoking cessation clinic or pharmacological therapy.At present, the quality of diagnosis, assessment and management of COPD is variable and does not always relate to the available knowledge about the disease and the burden it represents. The aims of this study were to assess the diagnostic accuracy of COPD registers in general practice and to evaluate guideline adherence in the treatment of stable COPD.MethodsRecruitment procedureAll practices in the Plymouth area were invited to take part. The first 13 practices to respond were included. Three North Devon practices agreed to participate.Project nurses (Respiratory Specialist Nurses trained and experienced in primary care management of COPD including spirometry) collected data from February 2005 to March 2006. Practice registers were electronically searched using diagnostic codes for COPD. Exclusion criteria were: serious co-morbidity affecting the patient's ability to take part or to perform spirometry, or inability to attend the surgery. Records of all patients on the COPD register were examined and those with coding errors or normal spirometry were excluded. The remaining suitable patients were invited to an appointment with the project nurse.Audit procedureThe South West Multicentre Research Ethics Committee confirmed that as a service evaluation, formal research ethics approval was not required for the audit. Patients were informed about the study and confidentiality issues. Patient consent was obtained to collect and analyse the data using an electronic consent form approved by the NHS information security and registration authority.Patient assessment was based on a custom written software package. Demographic and clinical data were entered by the project nurse (Table 1) and questionnaire data were entered by the patients. Spirometry was performed without bronchodilator therapy in all patients according to European Respiratory Society and American Thoracic Society standards [23]. The spirometers used were a MicroLab ML3500 (Plymouth) and a MicroLab ML3300 (North Devon). Reversibility testing was performed using 400–800 mcg salbutamol via a large volume spacer, with spirometry repeated after 15 minutes. In Plymouth, reversibility testing was carried out only if clinically indicated to separate asthma from COPD; in North Devon, reversibility testing was performed on all patients.Table 1Data gathered by the COPD assessment software (questionnaires not shown)Demographic dataWeight, height and body mass index (BMI)Spirometry results: FEV1 and FVC (litres and % of predicted) pre and post bronchodilatorNumber of exacerbations in previous yearNumber of antibiotics for respiratory tract infections in the previous 12 monthsNumber of oral steroid courses in the previous 12 monthsNumber of out of hours visits in the previous 12 monthsNumber of attendances of Accident and Emergency (A & E) in the previous 12 monthsNumber of hospital admissions in the previous 12 monthsNumber of bed days in the previous 12 monthsWhether patient had an x-ray at the time of diagnosis or in the previous 5 yearsVaccination status (pneumococcus and influenza)Current COPD medication: short and long acting bronchodilators, inhaled corticosteroids, mucolytics, other prescribed medicationsInhaler technique: good, moderate, poorUse of nebuliserSmoking history: age smoking started, date of cessation, average number of cigarettes per dayWhether patient had undergone smoking cessation treatmentOxygen assessment and therapy, cor pulmonale, cyanosisPulse oximetry valueAttendance of specialist services: respiratory specialist nurse/physiotherapy in the previous 2 years, chest clinic in the previous 5 years, pulmonary rehabilitation everAfter clinical assessments, patients completed on-screen questionnaires. One question was seen at a time, and each possible response was numbered. Patients selected their response and pressed the appropriate number on the keyboard.Questionnaires included the Medical Research Council (MRC) Dyspnoea Scale [24], the Clinical COPD Questionnaire (CCQ) [25] and the Lung Information Needs Questionnaire (LINQ) [22].Patients were also asked about their sputum production, (in order to assess the need for mucolytic therapy), and whether their symptoms were relieved by short-acting bronchodilators, (to help assess whether they were receiving adequate bronchodilator treatment).Following completion of clinical assessments and questionnaires, the program automatically determined the patient's recommended treatment according to the NICE guidelines. For example, inhaled steroids were recommended if FEV1 was less than 50% predicted and if the patient had had two or more exacerbations during the previous year. Recommendations for bronchodilator treatment were based on the nurse's clinical judgement and whether their current therapy relieved their symptoms rather than computer logic. Mucolytic therapy was recommended to be considered if the patient had a chronic productive cough.At the end of the assessment, the nurse reviewed the collected data and confirmed the diagnosis. The computer then produced two separate reports summarising the results of the assessments; a clinical report for the GP or practice nurse and a report in layman's terms for the patient.The COPD assessment softwareThe software was revised and improved during the process of the audit in accordance with feedback from patients, project nurses and primary care clinicians. Revisions included requesting data on treatment according to guidelines, shortening GP reports, and simplifying patient reports. As a result of this, there are varying numbers of patients in the statistical analyses.Diagnostic criteriaSpirometric results were interpreted according to the NICE recommendations [5]. If reversibility testing was applied, diagnosis was based on post-bronchodilator values. A diagnosis of restriction was given if the FEV1 (% predicted) was less than 80% and the ratio of FEV1 to forced vital capacity (FVC) was greater or equal to 0.7.Asthma was diagnosed on the basis of both spirometric and clinical features such as the patient's history and family history which were obtained from the patient and examination of their primary care records. Asthma was confirmed if a patient with airflow obstruction returned to normal spirometric values or if a large change in FEV1 (> 400 ml) was observed in response to bronchodilators. Some patients had clinical features of both asthma and COPD as described in the NICE guidelines.Data analysisData was transferred into SPSS (Version 14.0, SPSS Inc., Chicago IL). Descriptive statistics and cross-tables were used to evaluate the accuracy of diagnostic registers and guideline adherence in treatment. If applicable, t tests or chi square tests were used to analyse between-group differences (α = 0.05).ResultsCharacteristics of invited and excluded patientsOf the 841 patients invited for assessment, 619 were seen. Thirty-nine patients (6%) were unable to perform spirometry.To examine the possibility of selection bias, a sub-sample of 288 patients on the COPD register were examined in more detail. Of these patients, 47 (16%) were excluded as they were unable to attend the practice; the remaining 241 patients were invited. Of these, 43 (18%) patients declined. No differences in age and gender were seen between those who attended and those who declined (Table 2).Table 2Age and gender of North Devon patients who declined and who were seenDeclinedAttendedSignificanceAge66.4 (11.3)68.1 (10.0)t(241) = -0.997, p = 0.320Gender (Males)18/43 (42%)127/198 (64%)X2(1) = 0.27, p = 0.603Accuracy of diagnostic registersOf the 580 patients who underwent spirometry, 88 (15%) patients had normal values, 456 (79%) showed obstruction, and 36 (6%) showed restriction.Final diagnoses based on spirometric and clinical features are shown in Figure 1. Of 580 patients who had spirometry, 158 (27%) were found not to have COPD, as per the NICE guidelines. Of all 422 patients who received the final diagnosis of COPD, 25 patients (6%) had both asthma and COPD.Figure 1Final diagnoses in the Plymouth COPD audit sample.Reversibility testing in patients with airflow obstructionAirway obstruction was found in 456 patients and reversibility testing was undertaken in 232 (51%). In 140 patients reversibility testing was performed routinely (North Devon patients) and in 92 patients as clinically indicated to exclude asthma from COPD (Plymouth patients).Reversibility testing led to a change in diagnosis for 25/232 (11%) patients. Where reversibility testing was performed routinely, the diagnosis changed in 7/140 (5%), with five being changed from a diagnosis of obstruction to normal, and two changing from a diagnosis of obstruction to one of restriction. Where reversibility testing was performed as clinically indicated, diagnostic changes occurred in 18/92 (20%) patients, with 14 changing from a diagnosis of obstruction to normal, and four changing from a diagnosis of obstruction to restriction.As regards the magnitude of change in FEV1, 162 patients (70%) changed by up to 200 ml, 58 patients (25%) changed by 200–400 ml, and in 12 patients (5%) the change was larger than 400 ml.In patients who showed a small change of FEV1 (up to 200 ml), diagnosis was changed in 8/162 (5%). In patients with a change up to 400 ml, diagnosis was changed in 12/58 (21%), and in those with a change of more than 400 ml, diagnosis was changed in 5/12 (42%). In the large majority of cases the diagnosis was changed from COPD to asthma. Five of the 12 patients with more than 400 ml changes in FEV1 through reversibility testing met the criteria for asthma based on post bronchodilator readings alone.The severity grading of airflow obstruction based on pre-bronchodilator readings changed after bronchodilator in 41/232 (18%) patients. Thirty-one patients changed from moderate to mild disease, ten from severe to moderate. Three patients had lower spirometry readings after bronchodilators and in these cases their pre-bronchodilator readings were used to classify severity grading.Characteristics of patients with confirmed COPDThe cohort of 422 patients with confirmed COPD was examined further. The mean FEV1 was 1.25 (SD 0.44) litres and mean FEV1% predicted was 50.3% (SD 14.3). Based on the NICE guidelines, 227 (54%) patients had mild COPD, 159 (38%) patients had moderate COPD, and 36 (9%) patients had severe COPD.The mean (SD) age was 69.2 (8.7) years. Body mass index was low (< 18.5) in 26/420 (6%); 32% were normal and 62% were overweight (BMI > 25).One hundred and thirty-seven patients (33%) were current smokers with a mean consumption of 20 cigarettes per day and an exposure of 48 pack years; 276 (65%) were ex-smokers with a mean exposure of 43 pack years and ten patients (2%) had never smoked. Of the current smokers, 76% had been offered help to quit smoking, either with nicotine gum or patches, or by referral to a smoking cessation clinic.Exacerbations occurred in all grades of airflow obstruction. Patients with severe airflow obstruction had received more steroid courses in the previous year than patients with milder obstruction. Interestingly, healthcare consumption (out of hours visits, accident and emergency attendances, bed days spent in hospital) was not particularly skewed towards patients with severe airflow obstruction (Table 3). Respiratory failure or cor pulmonale was noted in five patients.Table 3Frequency of exacerbations, antibiotic and steroid courses and healthcare consumption in patients with confirmed COPDNumber in previous year of:Mild (N = 226)Moderate (N = 158)Severe (N = 36)All (N = 420)Exacerbations1.3 (1.7)1.3 (1.6)1.7 (2.3)1.4 (1.8)Antibiotics courses1.2 (1.6)1.3 (1.6)1.7 (2.3)1.3 (1.6)Steroid courses0.7 (1.3)0.7 (1.4)1.4 (2.2)0.8 (1.4)Out of hours visits0.1 (0.4)0.2 (0.5)0.2 (0.8)0.1 (0.5)Attended A&E0.1 (1.1)0.1 (0.5)0.2 (0.5)0.1 (0.9)Bed days0.5 (3.0)0.8 (4.0)0.7 (2.4)0.6 (3.3)Figure 2 shows counts of MRC dyspnoea scale scores in the COPD sample, for different degrees of severity. Results for the LINQ and the CCQ will be reported elsewhere.Figure 2Distribution of MRC dyspnoea score for different degrees of severity of airflow obstruction.In the previous two years, 93/422 (22%) patients had attended a consultant chest physician; 36/422 (9%) patients had seen a COPD specialist nurse and 3/422 (0.7%) had seen a respiratory specialist physiotherapist. In the previous five years, 187/384 (49%) of patients had had a chest x-ray, at time of diagnosis. Patients who had attended a consultant chest physician were similar to those who did not attend in respect of diagnosis and spirometry: mean FEV1% of predicted was 45.7% (SD 14.8) and 48.5% (SD 13.8) respectively (t = 1.68, p = 0.09), but had higher MRC dyspnoea scores: median 3.1 (IQR 1.3) and 2.7 (IQR 1.50), p = 0.002. No differences were noted between those attending and those not attending secondary care were noted with respect of age, smoking status, pack years or in the proportions that were on the recommended treatment with short and long acting anti-cholinergic, long acting beta-2-agonists inhaled steroids or mucolytics therapies.Management of COPD and compliance with guidelinesData on current or recommended treatment were available for 278 of the 422 patients diagnosed with COPD (Table 4).Table 4Current and recommended treatment in patients with confirmed COPD and proportion of patients in whom a treatment was recommended who were receiving that treatmentNo. currently receiving the treatmentNo. for whom the treatment was recommendedNo. receiving the treatment recommended for them(N = 278)(N = 278)(N = 278)SAAC124 (44.6%)109 (39.2%)59/109 (54%)LAB2 agonists124 (44.6%)80 (28.8%)37/80 (46%)LAAC50 (18.0%)53 (19.1%)12/53 (23%)Inhaled steroids167 (60.0%)48 (17.0%)39/48 (81%)Mucolytics9 (3%)144 (53.0%)6/144 (4%)Long acting bronchodilators were recommended for more patients than had received them and were therefore under prescribed. The NICE guidelines recommend that mucolytic therapy should be considered in patients with a chronic cough productive of sputum. Although 53% had a chronic productive cough, only 4% of these were receiving mucolytics. By contrast, 60% of patients were receiving inhaled steroids, of which only 23% met the indication of FEV1, less than 50% of predicted and two or more exacerbations per year [5]. Nine patients for whom inhaled steroids would be recommended had not received them, again highlighting the problem of inappropriate prescribing.Practices varied substantially in prescribing long acting bronchodilators. The proportion of patients prescribed long acting beta-2-agonists ranged between 23–56% in different practices, and between 9–25% for long acting anticholinergics.Prescribing outside of licensed indications was noted in six patients who were taking short acting anticholinergics and long acting anticholinergics therapy at the same time. Although long acting anticholinergics are only licensed for use in COPD, six patients who were found not to have COPD were on long acting anticholinergics.Vaccination status was recorded for immunisation against pneumococcus and influenza, both of which are recommended in COPD. Influenza vaccine was up to date in 346/398 (87%) and pneumococcal vaccination was up to date in 227/380 (60%).Data on attendance or recommendation to attend pulmonary rehabilitation were available for 372 COPD patients. Recommendations were based on the NICE guidelines which state that \"Pulmonary rehabilitation should be offered to all those who consider themselves functionally disabled by COPD (usually MRC dyspnoea scale score of three and above)\" [5].Pulmonary rehabilitation was not available in North Devon, but 54/134 (40%) North Devon patients had an MRC dyspnoea scale score of three or more. None of these patients had attended pulmonary rehabilitation. In Plymouth, 121/238 (51%) had MRC dyspnoea scale score of three or more and 80/238 patients (34%) were willing, suitable and able to take part. Of these, five had an MRCDS score of less than three and 7/80 (9%) had attended rehabilitation.DiscussionCOPD is an important disease that compromises patients' quality of life and creates a huge financial burden to the National Health Service. Our study confirms that disease management takes place largely in primary care. Guidelines suggest that an accurate diagnosis should be followed by effective treatments of stable disease and exacerbations of COPD.Diagnostic registers have inherent problems. According to the present findings, registers include large numbers without COPD. In this study, 27% of individuals listed as having COPD were eligible for reclassification of their disease following structured clinical assessment by a trained nurse. These findings confirm the problems previously observed with application and interpretation of spirometry in primary care.The application of reversibility testing is a controversial issue in the diagnosis of COPD in primary care [26]. Recent guidelines have suggested that reversibility testing should be used where clinically indicated to separate asthma from COPD [5,6], but there is a lack of good evidence to underpin these recommendations [26]. Reversibility testing records the change in FEV1 before and after bronchodilators and the magnitude of this change has been used to differentiate asthma from COPD [5,27].Reversibility testing was found to change the diagnosis from that made on pre-bronchodilator spirometry in 11% of cases. This finding demonstrates the benefits of judicious use of reversibility testing based on clinical need to exclude asthma as opposed to performing reversibility testing in all cases. This study confirms that performing pre-bronchodilator spirometry alone leads to overestimation of the prevalence and severity of COPD, with the potential to cause errors in treatment. The use of pre-bronchodilator spirometry alone cannot be recommended for diagnosis and severity assessment-spirometry in primary care should be done only after administration of bronchodilators. This approach is in keeping with the current GOLD guidelines, whereas the NICE guidelines do not specify the importance of post-bronchodilator readings.This study examined current treatment against treatment as recommended by the NICE guidelines. Under-prescribing with bronchodilators, particularly long acting agents was apparent. Long acting bronchodilators are known to improve lung function, exercise tolerance, symptoms, and quality of life, and to reduce exacerbations [6]. Furthermore, it is known that bronchodilators interact with other treatments to improve outcomes. The combination of fluticasone and salmeterol reduced decline in lung function over 3 years, [28] and outcomes in pulmonary rehabilitation were improved by tiotropium [29]. The reason why these drugs are not prescribed may be due to concerns over price, a lack of knowledge about the benefits of these medicines [19] or from an unjustified nihilistic approach to COPD management [30]. There was no evidence that this phenomenon was limited to primary care, those attending a consultant chest physician in the previous five years were no more likely to be treated according to guidelines.This study highlights an apparent over-treatment with inhaled steroids. Only a minority of those who received inhaled steroids met the NICE criteria. In some cases, treatment with steroids may once have been not only appropriate, but also effective in reducing exacerbations until they were no longer appeared necessary. Over-treatment suggests a waste of resources and puts patients at risk of adverse effects. These findings concur with Decramer et al. [15], who revealed that 49% of GPs prescribed inhaled steroids to all of their COPD patients. The role of mucolytics is debated [31], but despite the evidence-based recommendations in NICE that mucolytics should be considered in patients with a chronic productive cough, mucolytics are seldom prescribed to such patients.Quitting smoking improves patients' prognoses in COPD, and offering support to stop smoking reduces mortality and morbidity from COPD [1]. It is disappointing that a quarter of current smokers with COPD had not been offered practical help in terms of referral to smoking cessation service or drug therapy. While patients may not accurately report the help they have been offered, Rutschmann et al. [19] found that many physicians reported feeling uncomfortable giving smoking cessation advice, and a fifth of physicians were unaware that smoking cessation had a positive impact on life expectancy and disease progression.Pulmonary rehabilitation is highly effective and is a cost effective intervention recommended by national and international COPD guidelines. The finding in this study was striking-in certain geographic regions pulmonary rehabilitation was not available at all and where it was available, only 9% of eligible patients had attended. Identification of patients suitable for rehabilitation is an important component of COPD assessment, as currently the proven benefits of pulmonary rehabilitation are being denied to many patients.This study used a novel information technology system that provides a systematic and comprehensive assessment package for COPD, and facilitates the delivery of high-quality care according to guidelines. The system assesses clinical data and examines patient-centred outcomes such as quality of life and patients' perceived information needs. By providing reports to both GPs and patients it offers a new approach to COPD management in primary care. Management according to the guidelines is promoted with the potential to improve care. The system also has an educational function as GPs and nurses are given the opportunity to learn about COPD management. Given these advantages, use of the system has the potential to enable long-term improvement of COPD management. However, using a different IT-system for guideline-based management of COPD and asthma, Tierney et al. [32] reported no enhanced adherence to guidelines and no beneficial effects on patient-centred and clinical outcomes over a year. Research into the benefits of our system in terms of long-term improvement of clinical care and patient-centred outcomes is ongoing.This study has some limitations. Although the participating practices provided a spectrum of COPD services and varied widely in their size and socio-demographic features, they may not representative as they were all in Devon and were included on the basis of their interest in the project. Furthermore, as only patients who were able to attend primary care clinics were included, those most severely affected by their COPD and those with important co-morbidities may have been under-represented.The software system made recommendations for specific treatments based on guidelines and some recommendations were not definite statements that the treatment was required, but that the treatment should be considered, (for example mucolytics therapy). Thus the apparent under-prescribing in these situations is more difficult to interpret and may be appropriate use of therapy. A further limitation was that this was a cross-sectional study and did not assess the impact of recommendations on changing treatment or other outcomes. The actual decision by the GP to prescribe, the patients' compliance, response to treatment and duration were outside the scope of the study. The software system had not been formally validated in other studies.ConclusionThe present study represents one of the first studies to report the true levels of severity of COPD and the provision of appropriate treatments in primary care. The study demonstrates that compared to diagnoses made by expert nurses with the help of a standardised, guideline-based computer program, the diagnostic registers in primary care are inaccurate in 27% of cases. The role of reversibility testing was examined and it was found that pre-bronchodilator readings alone overestimated both prevalence and the severity of COPD. Reversibility testing was useful in detecting some cases where a diagnosis of asthma was considered. On this basis it is suggested that only post bronchodilator readings should be used routinely in primary care, but reversibility testing is appropriate in specific cases where asthma is suspected. The management of patients with COPD seldom followed guideline based recommendations in terms of drug treatment and pulmonary rehabilitation.In the future, computer-based assessment systems which provide management recommendations may facilitate optimal diagnosis and treatment for patients with COPD in primary care.Competing interestsDr Jones has received educational, research or travel grants from Glaxo Smith Kline; Astra Zeneca; Boehringer-Ingelheim; Pfizer; Ivax; Novartis and Altana. Dr Jones was a director of Patient Centred Software Ltd., the provider of the software used in this project.Authors' contributionsRJ and BS designed and supervised the study and contributed to the manuscript. MS analysed data and drafted the manuscript. MM recruited North Devon practices and carried out assessments in North Devon. DM acted as the project nurse and contributed to data preparation. All authors have read and approved the final manuscript.\n\nREFERENCES:\nNo References"
+}

batch_11/PMC2531201.json

@@ -0,0 +1,4 @@
+{
+    "id": "PMC2531201",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2531201\nAUTHORS: Bilal Chughtai, Christa Abraham, Daniel Finn, Stuart Rosenberg, Bharat Yarlagadda, Michael Perrotti\n\nABSTRACT:\nIntroduction. The aim of this study is to examine the feasibility of reducing postoperative hospital stay following open partial nephrectomy through the implementation of a goal directed clinical management pathway. Materials and Methods. A fast track clinical pathway for open partial nephrectomy was introduced in July 2006 at our institution. The pathway has daily goals and targets discharge for all patients on the 3rd postoperative day (POD). Defined goals are (1) ambulation and liquid diet on the evening of the operative day; (2) out of bed (OOB) at least 4 times on POD 1; (3) removal of Foley catheter on the morning of POD 2; (4) removal of Jackson Pratt drain on the afternoon of POD 2; (4) discharge to home on POD 3. Patients and family are instructed in the fast track protocol preoperatively. Demographic data, tumor size, length of stay, and complications were captured in a prospective database, and compared to a control group managed consecutively immediately preceding the institution of the fast track clinical pathway. Results. Data on 33 consecutive patients managed on the fast track clinical pathway was compared to that of 25 control patients. Twenty two (61%) out of 36 fast track patients and 4 (16%) out of 25 control patients achieved discharge on POD 3. Overall, fast track patients had a shorter hospital stay than controls (median, 3 versus 4 days; P = .012). Age (median, 55 versus 57 years), tumor size (median, 2.5 versus 2.5 cm), readmission within 30 days (5.5% versus 5.1%), and complications (10.2% versus 13.8%) were similar in the fast track patients and control, respectively. Conclusions. In the present series, a fast track clinical pathway after open partial nephrectomy reduced the postoperative length of hospital stay and did not appear to increase the postoperative complication rate.\n\nBODY:\n1. INTRODUCTIONRadical nephrectomy has considered the optimal surgical\napproach to the management of renal cancer [1]. Additional options include\nobservation, cryosurgery, and radiofrequency ablation [2, 3]. Management of the small renal mass requires multiple perspective\ndecision making on the part of the physician [4]. Recently, partial nephrectomy has been\nshown to have efficacy in the management of select patients with small renal\nmasses (4 cm or less) [5, 6]. Several investigators have reported\nan expanded use of this approach with success in patients with larger renal\nneoplasms (7 cm or less). Hospital stay\nafter partial nephrectomy is usually 5–8 days [7]. Factors limiting early discharge are\nusually pain, stress-induced major organ dysfunction (i.e., ileus, atelectasis),\ntradition, fatigue, pain, nausea, and morbidity [8, 9]. In other abdominal procedures, including\ncolon resection, lung transplant, and laparoscopic nephrectomy, the\nintroduction of a program comprised of optimized pain relief using nonsteroidal\nanti-inflammatories for analgesia enforced oral nutrition and mobilization, and\nrevision of traditional care principles has reduced hospital stay from 5–8 days to 220 133\ndays in some studies [10, 11]. The concept of fast-track (FT) surgery\nhas recently attracted more interest, but has not yet been applied in patients\nundergoing partial nephrectomy. The purpose of this study was to investigate\nthe postoperative course before and after the introduction of a fast track program\nin patients undergoing open partial nephrectomies. We sought to determine\nwhether the use of this fast track program might decrease length of hospital\nstay without sacrificing outcomes.2. MATERIALS AND METHODSA fast\ntrack clinical pathway for open partial nephrectomy was introduced at our\ninstitution in July 2006. The pathway\nhas an established management protocol (Table 1). All patients undergoing open\npartial nephrectomy from July 2006 thus far at our institution were managed by\nthe fast track protocol, and comprise the study cohort. Patients undergoing laparoscopic partial\nnephrectomy and robotic partial nephrectomy were not included. In the event that the decision was made\nintraoperatively to perform complete nephrectomy, patients were included on an\nintention-to-treat basis. Demographic\ndata, tumor size, blood loss, transfusion, final pathology, margin status, length\nof hospital stay, and complications were captured in a prospective database,\nand compared to a control group managed consecutively immediately preceding the\ninstitution of the fast track clinical pathway.\nAll operations were performed for a renal tumor less than 7 cm in\ngreatest diameter by the same surgical team.2.1. Preoperative preparationPatients\nwere admitted to the hospital on the day of surgery and performed all\npreparation on an outpatient basis. All\npatients were counseled preoperatively regarding the target goals outlined in\nTable 1. Aspirin and nonsteroidal\nanti-inflammatory drugs (NSAIDs) were stopped 10–14 days prior to\nsurgery. Coumadin was stopped 5 days\npreoperatively and stat protime/partial thromboplastin time obtained on the morning of surgery\nto confirm acceptable value. All patients received 3 bisacodyl\ntablets and 1 bottle of magnesium citrate at noon on the day prior to surgery. All patients\nreceived erythromycin and neomycin antibiotic per oral (PO),\n500 mg (milligram) tab, at 3 pm, 6 pm, and 9 pm on the day prior to surgery. All patients were instructed to eat lightly\nand orally hydrate on the day prior to surgery.2.2. IntraoperativeThis\nreport is restricted to patients undergoing open partial nephrectomy on an\nintention to treat basis. General anesthesia, oral gastric tube (removed at\ncase conclusion), and Foley catheter were used in all cases. Compression pneumatic stockings were placed\nas soon as the patient moved from the stretcher onto the operating table. The retroperitoneal flank approach was\nused. All patients were positioned with\nthe bean bag, with legs straight, hyper-extended with the kidney rest raised\nmaximally. Before draping, the surgeon\nmarked the posterior axillary line (PAL),\nthe anterior axillary line (AAL), the lateral border of the rectus muscle, and\nthe course of the 10th, 11th, and 12th ribs. Incision was made from the PAL to the AAL in the course of the 11th rib using the cautery on pure cut (setting of 30). The distal tip of the 11th rib was removed in the standard manner.\nAn extra-pleural/extra-peritoneal approach to the kidney was used. The\nkidney was explored, and vessel loop passed to tag the ureter, renal artery,\nand renal vein. A double loop was passed around the vein for subsequent\nocclusion. Patients received mannitol\n12.5 gm (gram) IV (intravenous) bolus prior to manipulation of the renal vessels\nfollowed by 5 gm/hour continuous infusion for the remainder of the operation.\nRenal artery was clamped in all cases and the kidney cooled. The renal vein was\nselectively occluded as required to provide a bloodless operative field. The\ncollecting system was closed with 3–0 monocryl on SH\nneedle in all cases. Renal arteries and venules were oversewn in\nfigure of eight fashions\nwith 3–0 monocryl on SH\nneedle. Prior to the removal of the renal artery clamp, the kidney was reconstructed essentially\nobliterating the resection defect utilizing 0-Chromic suture on CT needle in\nhorizontal mattress\nfashion. This resulted in a reniform shape approximation in nearly all cases. The rib bed\nand skin were infiltrated with 0.25% marcaine (30 ml (milliliters)). The skin\nwound was closed in a subcuticular (3–0 monocryl). A #7 Jackson-Pratt drain was placed in all\ncases. In no case was a ureteral stent\nplaced.2.3. PostoperativeOn POD 0 (postoperative day), on the evening of surgery,\npatients received celecoxib 200 mg per oral\nin the postanesthesia care unit (PACU) with sip when awake, and daily thereafter (Table 1). Morphine sulfate was administered IV at 2 hour\nintervals as needed. Metoclopramide was\nadministered IV 10 mg every 6 hours. Famotidine was administered 20 mg IV every 12 hours. On the day of surgery, patients ambulated and\nwere encouraged to take liquids by mouth.\nOn POD 1, diet was advanced to tray of clears, and oral pain medication\nadministered (hydrocodone/acetominophen 5–10 mg/500 mg every 4 hours as\nneeded). On day 2, the Foley catheter\nwas removed at 7 am and regular diet initiated.\nPatients received milk of magnesia 30 cc PO at 8 am and a repeat dose in 4 hours.\nJackson-Pratt drain fluid was sent to the laboratory for creatinine\nmeasurement and if equal to serum creatinine level, the Jackson-Pratt drain was\nremoved. The patient was assessed and\ndischarged to home on POD 3 if appropriate.3. RESULTSA total of 33 patients were managed\nby fast track and compared to 25 control patients (Table 2). The estimated\nblood loss, transfusion rate, tumor size, pathology, and complication rate were\nsimilar between groups. There was, however, a significant difference in the\nlength of hospital stay observed between groups. Of 25 control patients, 4 (16%) achieved\ndischarge to home in <3 days compared to 22 (67%) of the 33 patients\nmanaged in the fast track program.\nOverall, fast track patients had a shorter hospital stay compared to\ncontrols (median, 3 days versus 4 days; P = .012). Of the 11 patients in\nthe fast track cohort who were discharged after the third postoperative day,\nthis was due to poor ambulation/inadequate pain control (n = 5), abdominal\nbloating (n = 3), multiple co-morbidities (n = 2), and respiratory distress\npostoperatively requiring ICU care (n = 1).Complications are worth noting to\ndetermine whether the fast track approach was harmful in any way to the study\ncohort. In the control cohort, there was\n1 patient with respiratory distress requiring ICU admission, 3 patients\nreceived blood transfusion, there were 2 conversions to complete nephrectomy,\nand 1 positive surgical margin. In the\nfast track cohort there was 1 patient with respiratory distress requiring ICU\nadmission, 2 patients required blood transfusion, there was one conversion to\ntotal nephrectomy, 1 postoperative bleed (gross hematuria) requiring selective\narterial embolization, and 1 urine leak requiring percutaneous drainage and\nureteral stent placement.The percentage of patients with\nmalignancy increased in the fast track cohort compared to control (85% versus\n76%). This may represent improved\npreoperative assessment.4. DISCUSSIONSince 1950 in the United States, there has been a\n126% increase in the incidence of renal cancer [7, 8]. Although there has been an increase\nin all stages of renal cancer including advanced cases (i.e., regional\nextension, distant metastases), there has been the greatest increase in those\ndiscovered incidentally [8, 12]. In the early 1970s, approximately\n10% of tumors were detected incidentally compared with 61% in 1988 [8].Previous studies of other types of major surgery have shown\nthat a combined effort comprising intensive preoperative information, effective\npostoperative pain relief and enforced mobilization, and early enteral\nnutrition can accelerate postoperative recovery and decrease hospital stay [11, 13].Investigators have recently illustrated that in elderly\nhigh-risk patients undergoing colonic resection, mean hospital stay could be\nreduced to 2-3 days [11]. In another group of high risk\npatients undergoing open aortic surgery, mean hospital length of stay was\nreduced from a mean of 9 days to 5 days [14–16]. In a study by Harinath et al., a\ndecrease in length of stay was observed from 5 to 4 days for ileal pouch-anal\nanastomosis [11]. The concept of fast track has also\nbeen applied to infants and children [17]. In the urologic literature, after\nradical prostatectomy, median hospital stay in 252 consecutive patients was\nreduced to 1 day [13]. Specifically with kidney surgery,\npostoperative hospital stay after open nephrectomy was reduced to 4 days [18]. With laparoscopy, hospital stay has\nbeen reduced to 2 days with an FT rehabilitation program [19].In this study, with the introduction of a fast-track program,\nopen partial nephrectomy hospital stay was decreased to 3 days, compared to 4\ndays before implementation of the program. Sixty six per cent of patients\nachieved a target discharge on day 3 or less.\nNotably, both groups did have similar characteristics as demonstrated in\nTable 2. The estimated blood loss, transfusion rate, tumor size, pathology, and\ncomplication rate were similar between groups. We suspect that based upon our\ndata the main contributing factors responsible for the decrease in hospital\nstay was a clear protocol of expectations at each stage of the recovery\nperiod. This was accomplished in the\npresent series without an apparent increase in complication rate. Most notably, fast track did not lead to an\nincrease in readmissions.It is\nimpossible to discern exactly which components of our protocol are “more\nessential” than others, and this would require selective application in future\ninvestigations. In addition, we have no proof from the present\ninvestigation that the fast track protocol is advantageous to the\npatient. The purpose of the present study was to assess the feasibility\nof such an approach and we conclude that such an approach is feasible.\nUltimately, the patients in this study decided their discharge date.\nMost patients were eager to receive discharge to home as soon as they are\nmedically safe. One patient in the fast track protocol was discharged to\nhome at her request on POD 2. She expressed regret at doing so at\nher first postoperative visit.Our readmissions\nto the hospital were as follows. One (3.0%) patient had returned to hospital for postoperative\nhemorrhage resulting in gross hematuria. This patient was managed successfully\nwith embolization. One (3.0%) patient had returned to hospital\nforurinoma. This was managed successfully with indwelling ureteral\nstent for 6 weeks and transient percutaneous drainage of urinoma. Both patients had\ncomplex resections, and it is unlikely that fast track management resulted\nin return to hospital.5. CONCLUSIONSIn the\npresent investigation, a fast track clinical pathway after open partial\nnephrectomy reduced the postoperative length of hospital stay and did not\nappear to increase the postoperative complication rate.\n\nREFERENCES:\n1. KunkleDAEglestonBLUzzoRGExcise, ablate or observe: the small renal mass dilemma—a meta-analysis and reviewThe Journal of Urology200817941227123418280512\n2. BandiGHedicanSPNakadaSYCurrent practice patterns in the use of ablation technology for the management of small renal masses at academic centers in the United StatesUrology200871111311718242377\n3. MatuszewskiMKrajkaKRadio-frequency ablation: new technology for palliative treatment of hematuria in disseminated renal cell carcinomaScandinavian Journal of Urology and Nephrology200741656356417853039\n4. PerrottiMBadgerWJMcleodDPraterSMoranMEDoes laparoscopy beget underuse of partial nephrectomy for T1 renal masses? Competing treatment decision pathways may influence utilizationJournal of Endourology200721101223122817949330\n5. AronMGillISPartial nephrectomy—why, when, how...?The Journal of Urology2008179381181218221968\n6. RiggsSBKlatteTBelldegrunASUpdate on partial nephrectomy and novel techniquesUrologic Oncology: Seminars and Original Investigations200725652052218047964\n7. MerseburgerASKuczykMAChanging concepts in the surgery of renal cell carcinomaWorld Journal of Urology200826212713318265990\n8. RamírezMLEvansCPCurrent management of small renal massesThe Canadian Journal of Urology200714supplement 1394718163944\n9. FiroozfardBChristensenTHBendixenANordlingJKehletHNephrectomy in denmark 2002–2005Ugeskrift for Laeger2006168151526152816640972\n10. KarivYDelaneyCPSenagoreAJClinical outcomes and cost analysis of a “fast track” postoperative care pathway for ileal pouch-anal anastomosis: a case control studyDiseases of the Colon & Rectum200750213714617186427\n11. HarinathGSomasekarKHarayPNThe effectiveness of new criteria for colorectal fast track clinicsColorectal Disease20024211511712780633\n12. PorpigliaFVolpeABilliaMScarpaRMLaparoscopic versus open partial nephrectomy: analysis of the current literatureEuropean Urology200853473274318222599\n13. HeinzerHHeuerRNordenflychtOVFast-track surgery in radical retropubic prostatectomy. First experiences with a comprehensive program to enhance postoperative convalescenceDer Urologe A2005441112871294\n14. MurphyMARichardsTAtkinsonCPerkinsJHandsLJFast track open aortic surgery: reduced post operative stay with a goal directed pathwayEuropean Journal of Vascular and Endovascular Surgery2008343274278\n15. Marrocco-TrischittaMMMelissanoGChiesaRLetter to the editor regarding “Fast track open aortic surgery: reduced post operative stay with a goal directed pathway”European Journal of Vascular and Endovascular Surgery2008352251 pages18065246\n16. MuehlingBMHalterGLangGProspective randomized controlled trial to evaluate “fast-track” elective open infrarenal aneurysm repairLangenbeck's Archives of Surgery20083933281287\n17. ReismannMvon KampenMLaupichlerBSuempelmannRSchmidtAIUreBMFast-track surgery in infants and childrenJournal of Pediatric Surgery200742123423817208572\n18. FiroozfardBChristensenTKristensenJKMogensenSKehletHFast-track open transperitoneal nephrectomyScandinavian Journal of Urology and Nephrology200337430530812944188\n19. RecartADucheneDWhitePFThomasTJohnsonDBCadedduJAEfficacy and safety of fast-track recovery strategy for patients undergoing laparoscopic nephrectomyJournal of Endourology200519101165116916359206"
+}

batch_11/PMC2532487.json

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+{
+    "id": "PMC2532487",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2532487\nAUTHORS: Y. Lynn Wang, Qi Miao\n\nABSTRACT:\nThe role of PPARγ in tumorigenesis is controversial. In this article, we review and analyze literature from the past decade that highlights the potential proneoplastic activity of PPARγ. We discuss the following five aspects of the nuclear hormone receptor and its agonists: (1) relative expression of PPARγ in human tumor versus normal tissues; (2) receptor-dependent proneoplastic effects; (3) impact of PPARγ and its agonists on tumors in animal models; (4) clinical trials of thiazolidinediones (TZDs) in human malignancies; (5) TZDs as chemopreventive agents in epidemiology studies. The focus is placed on the most relevant in vivo animal models and human data. In vitro cell line studies are included only when the effects are shown to be dependent on the PPARγ receptor.\n\nBODY:\n1. INTRODUCTIONPPARγ is a nuclear hormone receptor that requires\nligand binding for activation. In 1995, it\nwas discovered that PPARγ is the molecular target of thiazolidinediones\n(TZDs, [1]), a class of synthetic\ncompounds that are effective for the treatment of type 2 diabetes. This\ndiscovery spurred great interest in these agents, as well as in the receptor. Besides its function as an insulin sensitizer\nin diabetes, PPARγ was found to have a variety of roles in immunoregulation,\natherosclerosis, angiogenesis, and tumorigenesis.With regards to carcinogenesis,\ndebate continues as to whether PPARγ is pro- or antineoplastic, despite very active\nresearch over the past few years. At the cellular level, PPARγ was found to be involved in cancer cell\nsurvival/apoptosis, proliferation, and differentiation. While the apoptotic functions\nof PPARγ and its agonists are addressed by others in\nthis special issue, we will conduct a critical review of the literature that\nsuggests that PPARγ has a prosurvival activity. The review is mainly focused on data derived\nfrom in vivo models and/or human studies. In vitro cell line-based studies are\nincluded only when the effects are shown to be dependent on the PPARγ receptor.One important\nlesson learned from the past several years of research is that effects observed\nwith agonists of PPARγ are not necessarily intrinsic effects of the\nnuclear hormone receptor. In tumor cell survival, the proapoptotic activities of\nPPARγ agonists in various tumors act through both\nreceptor-dependent and receptor-independent mechanisms. When reviewing the\nliterature, we advise that the readers carefully consider the following to\ndistinguish drugs or TZDs versus receptor effects: (1) are high or low doses\nused in the studies? High or low doses\nshould be defined with respect to EC50 of glitazones in the PPARγ transactivation assays (Table 1) or plasma\nconcentrations that can be reached in humans (Table 2). Effects observed with\nhigh concentrations may not be relevant due to toxicities of certain TZDs, such\nas hepatotoxicity of troglitazone and potential cardiotoxicity of rosiglitazone\n(see below). (2) Are multiple pharmacological agents used? If a pharmacological\napproach is the only one used, claims of a receptor-dependent effect require\ndemonstration with agonists of different chemical structures, such as TZDs,\ntyrosine analogues, 15-Deoxy-Δ12,14-PGJ2 (15d-PGJ2),\nand so forth. Beware that 15d-PGJ2 possesses many PPARγ-independent activities, including inhibition\nof the NFκB pathway, that are known to have\nprosurvival and anti-inflammatory properties, as well as other effects [2–4]. (3) Are any antagonists\nincluded in the study? Do antagonists GW9662 or T0070907 block or reverse the\nobserved effects? (4) Are there any experiments in the study utilizing a genetic\napproach to confirm the pharmacological findings? Does the study involve cell lines or primary\ncells that contain or lack PPARγ, preferably in the same genetic background? For\nthose cell lines with endogenous PPARγ, is the siRNA, shRNA or dominant negative form\nof PPARγ used to reduce the levels of the receptor? Are\nspecific effects of the receptor diminished by such reduction? For readers'\nconvenience, these questions are summarized in Table 3.2. EXPRESSION OF PPARγ IN HUMAN TUMOR\nVERSUS NORMAL TISSUESIt is generally believed that expression of\na gene in a particular tissue suggests that the activity of the encoded protein\nis required for certain cellular functions of that tissue. In so far as cancers\nare concerned, the general rule is that oncogenes are overexpressed due to\ndysregulation, and tumor suppressor genes are underexpressed or absent due to\nmutations or deletions. In order to clarify the roles of the PPARγ receptor,\nit would be informative to review the expression levels of PPARγ in\ntumors with respect to their normal tissue counterparts. In this article, expression\ndata from tumor cell lines are not included.A review of the current literature on human\ncancers showed that expression levels of PPARγ mRNA and protein are generally higher in neoplastic\ntissues than their normal counterparts (summarized in Table 4). The most convincing data came from a large\nstudy of prostate cancer that included 156 patients with prostate cancer (PC),\n15 with less aggressive prostatic intraepithelial neoplasia (PIN), 20 with\nbenign prostatic hyperplasia, and 12 normal prostate tissues. In this study, a high level of PPARγ expression,\nby immunohistochemistry, is observed in PC and PIN cases in comparison to low\nor no expression in the benign hyperplasia and normal tissues. The results were\nconfirmed at the mRNA level with RT-PCR on a few cases from each category of\nthe malignant and benign conditions [13]. A large study of 126 renal cell carcinomas\nalso showed significantly more extensive and intensive PPARγ staining\nin tumor epithelium compared to the average staining levels seen in 20 normal\ntissues [14]. Similarly, in 22 patients with nonsmall\ncell lung carcinoma, higher levels of PPARγ are\nexpressed in tumor cells than in the surrounding normal tissue, as determined\nby immunohistochemical staining. In addition, higher expression levels in tumor\ncells are confirmed by Western blotting hybridization, using homogenized tissue\nsamples [15]. In hepatocellular carcinoma, immunostaining\nalso demonstrates that PPARγ is\noverexpressed in all of 20 carcinoma tissues but not in normal hepatocytes [16]. For squamous cell carcinoma, 20 cases of\nprimary tumor and six cases of lymph node metastasis were demonstrated to have\nincreased PPARγ protein\nexpression compared to normal tongue tissue [17]. Infiltrating adenocarcinoma of the breast\nalso expresses higher nuclear staining of PPARγ compared\nto normal ductal epithelial cells by immunohistochemical analysis. However,\nonly one of the three cases was shown [18]. For papillary thyroid carcinoma, six\npatients were studied to determine PPARγ mRNA\nexpression using reverse transcription PCR. The message was found in three of\nsix tumor tissues while the corresponding normal tissues do not express PPARγ [19].Follicular thyroid carcinoma, a less common\nhistological subtype of thyroid cancer, is characterized by a chromosomal\ntranslocation t(2;3) that results in a fusion between paired box gene 8 on\nchromosome 2 and PPARγ on\nchromosome 3 (PAX8-PPARγ). The fusion protein was initially thought to\nfunction as a dominant-negative inhibitor of the wild-type PPARγ protein\n[28]. However, a recent microarray study revealed\nthat (1) PPARγ transcript\nlevels in all seven cases of PAX8-PPARγ-containing\nfollicular carcinomas are more than 10-fold higher than normal thyroid tissues,\nas determined by both microarray and quantitative RT-PCR analyses; (2) the\nexpression profile of the fusion-positive follicular carcinomas shows induction\nof genes that are involved in fatty acid, amino acid, and glucose metabolic\npathways. Interestingly, many of the upregulated genes are known\ntranscriptional targets of the wild-type receptor, suggesting that the PAX8-PPARγ\nfusion protein functions similarly to wild-type PPARγ, rather\nthan antagonizing its activity. (3) Using cell lines transfected with PPARγ or the\nfusion protein, it is shown that expression of some genes, including angiogenic\nfactors PGF and ANGPTL4, is specifically upregulated by the fusion protein, particularly\nin the absence of ligand, indicating that the fusion protein is constitutively active.\nTaken together, these experimental data suggest that the translocation enhances\nthe function of PPARγ in a way\nthat contributes to the development or progression of follicular carcinoma of the\nthyroid [29].Upregulation of PPARγ has\nbeen demonstrated during tumor progression. Mueller et al. have found\nsignificant PPARγ staining\nin six cases of metastatic breast adenocarcinoma. In cell lines established\nfrom the primary and metastatic tumors of one of these patients, significantly higher\namounts of PPARγ transcript\nare shown in the cell line derived from the metastatic tumor [20]. In ovarian cancer, intensity and location\nof PPARγ immunostaining\nwere examined in 28 carcinoma cases along with 28 normal, benign or borderline cases.\nTwenty six of 28 carcinomas showed strongly positive PPARγ staining\ncompared to 2 weak-staining cases in the control group. Moreover, it is noted\nthat PPARγ staining\nwas predominantly nuclear in grade 2 or 3 tumors, as compared to a predominantly\ncytoplasmic staining pattern in grade 1 tumors [21]. Similar findings were made in transitional\ncell carcinoma of urinary bladder. Whereas no significant PPARγ immunoreactivity\nwas observed in 20 normal tissues, elevated PPARγ was\nfound in 168 tumors. Furthermore, the intensity of staining increased as the\nhistological grade increased from G1 to G3 and the tumor stage increased from\nearly (pT1 or lower) to advanced (stage 2 or higher) [22].A recent large study of 129 cases of\npancreatic ductal adenocarcinoma convincingly showed by array-based gene\nprofiling that expression of PPARγ in the\ntumor cells is ~7 fold higher than that in the normal ductal epithelia. This\nfinding was confirmed with immunohistochemical analysis of the tissue sections.\nNormal ductal epithelia showed insignificant staining for PPARγ. An\nearly lesion, intraepithelial neoplasia showed occasional PPARγ expression\nwhereas more than 70% of invasive pancreatic carcinoma demonstrated weak to\nstrong expression. Statistical analysis indeed revealed that expression of PPARγ correlates\nwith high tumor stage and higher tumor histological grade. More strikingly,\nexpression of PPARγ in pancreatic\ncancer is shown, by multivariant survival analysis, to be a significant\nprognostic indicator for shortened patient survival [23].In parallel\nto the above literature, levels of PPARγ mRNA\nfound in several well- or poorly-differentiated colorectal adenocarcinomas,\nwere similar to normal tissues [24]. Another group also found that the PPARγ immunostaining\nin well-, moderately-, or poorly-differentiated gastric adenocarcinomas is\ncomparable to that in noncancerous tissue adjacent to the tumor [25]. In liposarcomas, PPARγ\ntranscript levels are similar to that of the adipose tissue [26]. In adrenal glands, there is, again, no significant difference in mRNA\nexpression among cases of carcinoma, adenoma, and normal tissues [27]. Notably, at the time of composition of\nthis manuscript, we have not yet found any reports stating that PPARγ expression\nis downregulated or absent in human tumor versus normal tissues (Table 4).The next\nquestion is whether or not the PPARγ\nexpressed in tumor tissues is functional. Are ligands of PPARγ\npresent in the tumor tissues? A thorough and up to date literature search yielded\nfew results. The English abstract of a\nstudy published in a foreign language stated that there was no significant\ndifference in 15d-PGJ2 concentration between gastric cancer tissues\nand controls [30].\nAn earlier study showed that 15d-PGJ2 promotes the proliferation of HCA-7, a cyclooxygenase 2 (COX-2)-containing\ncolon cancer cell line at nanomolar concentrations. Further characterization by HPLC and mass\nspectrometry identified PGJ2, a chemical precursor of 15d-PGJ2 in the culture medium of HCA-7 cells [31]. COX-2 is a key enzyme in the\nbiochemical pathway that leads to the formation of cyclopentenone\nprostaglandins including 15d-PGJ2.\nOverexpression of COX-2 has been documented in many cancer types and contributes\nto tumor growth [32]. Overall, these few and somewhat circumstantial evidences\nsuggest that 15d-PGJ2 might be present in the tumor tissues.Does PPARγ lose\nor gain abnormal functions through mutations other than PAX8-PPARγ\ntranslocation? A large survey of human tumor samples and cancer cell lines does\nnot support such a notion. The exon 3 and 5 mutations, once reported in\nsporadic colon cancers [33], were not present in nearly 400 cell lines\nand primary tumor samples including lung, breast, prostate, colon cancers, and\nleukemias [34].Taken\ntogether, several lines of evidence regarding PPARγ expression\nsuggest a positive contributive role of the receptor in the development,\nmaintenance, or progression of human malignancies: (1) PPARγ is\noverexpressed in the vast majority of cancers. (2) In several types of cancer, PPARγ expression\nis further increased during tumor progression. (3) The oncogenic fusion PAX8-PPARγ results\nin PPARγ overexpression\nand upregulation of a similar profile of transcriptional targets as the\nwild-type protein. (4) Expression of PPARγ in\npancreatic cancer is associated with shorter survival.3. RECEPTOR-DEPENDENT PRONEOPLASTIC\nEFFECTS OF PPARγ\nIs there also\ncellular-level evidence suggesting that PPARγ promotes tumors? Most studies, especially\nthose employing high doses of TZDs, suggest that PPARγ agonists have antitumor activities through inhibition\nof cell proliferation or induction of apoptosis or differentiation. However, receptor-independent pathways are\ninvolved in most of the cases (reviewed elsewhere in this special issue). Then what does the receptor by itself do in\ntumors?Schaefer et al. showed that inhibition of PPARγ induces apoptosis of hepatocellular carcinoma\ncells (HCCs) by preventing their adhesion to the extracellular matrix,\nsuggesting that the activity of PPARγ is required for HCC cells to adhere and\nsurvive [16]. In that study, those\nparticular effects were shown to be receptor-dependent. Loss of cell adhesion\nrequires almost complete loss of PPARγ activity achieved by either PPARγ-targeting siRNA or PPARγ inhibitor T0070907. In addition, T0070907\ncauses cell death at concentrations far lower than those needed for PPARγ agonists rosiglitazone and troglitazone.\nTogether, the data suggest that PPARγ functions to promote tumor cell adhesion and\nsurvival in HCC cells. In line with this notion, the promoter region of\nhepatocyte growth factor contains a functional PPAR response element (PPRE) that\nmediates its transcriptional upregulation by PPARγ.\nThe growth factor plays an essential role in liver growth during embryonic\ndevelopment, as well as in maintenance and renewal of cells in various organs including liver, lung, and kidney, in\nadulthood [35].Our laboratory studied\nhuman anaplastic large T-cell lymphomas, a common form of large cell lymphoma\nin the pediatric population. We first demonstrated with immunohistochemical\nstaining that PPARγ is expressed in the malignant cells of the lymphoma\ntissues [36]. We then tested the effect of\nPPARγ activation in cell lines established from\npatients with this lymphoma. A pair of cell lines, Karpas 299 and SUP-M2 that, respectively,\ncontain and lack endogenous PPARγ were selected to address the receptor-dependency\nissue. Additionally, only low ligand concentrations were used, following\ninitial dose titration, to minimize any off-target effects. Using this system,\nwe have found that low doses of PPARγ agonists do not affect cell survival under\nnormal conditions. When cell death was induced by nutrient deprivation through\nserum withdrawal, activation of the receptor with low doses of rosiglitazone\n(0.5–2 μM) attenuated cell death, as compared to drug\nvehicle-treated cells. This result was reproducible with low doses of GW7845\n(0.5–2 μM) and 15d-PGJ2(0.5–1 μM). The effect occurred only in PPARγ-containing Karpas 299 cells but not in PPARγ-lacking SUP-M2 cells. Moreover, reducing PPARγ in Karpas 299 cells with siRNA diminished the\nprosurvival effect of the receptor. Furthermore, we showed that the prosurvival\neffect is mediated through PPARγ-dependent cellular metabolic changes,\nincluding increased cellular ATP levels, stabilized mitochondrial membrane\npotential, and reduced reactive oxygen species (ROS) production that each favor\ncell survival. PPARγ does so through coordinated regulation of the expression\nof ROS metabolic enzymes, including the p67 subunit of NADPH oxidase, uncoupling\nprotein 2 (UCP2), and manganese superoxide dismutase (Mn-SOD) at both mRNA and\nprotein levels that lead to ROS limitation. Lastly, we showed that stable\ntransfection of PPARγ into SUP-M2 cells not only improved cell\nsurvival, but also suppressed ROS accumulation during serum starvation. These\ngenetic manipulations have provided definitive evidence that PPARγ promotes lymphoma cell survival under\nconditions of nutrient deprivation.Our group has also\nmade similar findings in a murine cellular model [37, 38]. FL5.12 is a murine lymphocytic\ncell line that requires interleukin-3 (IL-3) for survival and proliferation. This cell\nline has been extensively used to characterize tumor cell metabolism [39]. FL5.12 cells express little\nPPARγ, but are killed by high concentrations of PPARγ agonists, 15d-PGJ2 (≥10 μM) and ciglitazone (≥80 μM). In an FL5.12 cell line stably-transfected\nwith PPARγ, low doses of PPARγ agonist do not affect cell viability under\nnormal conditions. However, when cells are induced to die by IL-3 withdrawal,\nlow doses of ciglitazone (10 μM) and rosiglitazone (0.05–2 μM) improved survival in only PPARγ-containing cells. Improved cell survival is\nalso accompanied by stabilized mitochondria and reduced ROS. Moreover, ATP\nproduction is required for PPARγ to exert its prosurvival effect. In this\nsystem, expression of a different panel of ROS metabolic enzymes including\ncatalase, and Cu/Zn-SOD are\ninvolved in reduction of the cellular levels of ROS. Functional PPRE sequences were\nshown to be present in the promoter regions of these two genes, suggesting that\nthe upregulation of their expression could be directly regulated by PPARγ [40–42]. Taken together, data from\nboth human and murine cell line studies suggest that PPARγ promotes tumor cell survival under conditions\nof nutrient/growth factor deprivation, and that the effect is not limited to a\nparticular system. The mechanism by which PPARγ increases cell survival is diagrammed in\nFigure 1 (Also see below).In support of the\nprosurvival activity of PPARγ in T-cell malignancies, Ferreira-Silva et al. very\nrecently showed that RNAi-mediated silencing of PPARγ in Jurkat T-cells caused increased DNA\nfragmentation and apoptosis as well as G2/M cell cycle arrest, arguing that the\nreceptor, proper, promotes the viability of the tumor cells [43].In parallel to\nthese findings in tumors, the prosurvival activity of PPARγ has been well documented in certain nonneoplastic\npathological conditions, especially ischemia-reperfusion injury in\nnutrient-sensitive tissues such as brain, heart and kidney [44–51]. Irreversible damage that results from\nprolonged ischemia causes stroke, and myocardial and kidney infarction. At the cellular\nlevel, cell death occurs as a result of nutrient deprivation and inflammatory\nresponses that involve the actions of proinflammatory cytokines, chemokines and\ntranscriptional factors. In addition,\nincreased production of ROS plays an important role in causing damage to macromolecules\nand eventual cell death [52]. A recent study using a rat\nmodel of cerebral focal ischemia has shown that expression of PPARγ mRNA and protein is upregulated in the areas\nadjacent to infarct caused by middle cerebral artery occlusion [46]. Administration of glitazones\nprior to, at the time of, or shortly after ischemia induction causes an\nincrease in DNA binding of the receptor. This is accompanied by a decrease in the\nexpression of a number of inflammatory genes, along with an increase in the\nexpression of antioxidant enzymes including catalase and Cu/Zn-SOD [44–47]. Consequently, these changes\nlead to limited cell demise, which eventually results in significantly reduced infarct\nsize. This process apparently works through a PPARγ-dependent mechanism, as GW9662 can block these\neffects of TZDs in animals [47]. Another PPARγ antagonist, T0070907, even increases the infarction\nsize, both in the presence and absence of PPARγ ligands [46].In light of both\nthese findings and the overexpression of PPARγ in many cancers, it is reasonable to\nhypothesize that the function of PPARγ in cancer is to confer a survival advantage\nupon the malignant cells, allowing them to survive in an adverse environment.\nAs a result of fast growth, the center of a three dimensional tumor mass is\noften deprived of oxygen, growth factors, glucose, and other nutrients due to\nexcessive demand and insufficient vascularization. However, cancer cells\npossess remarkable tolerance and are able to survive despite the adverse\nconditions [53, 54]. Besides increasing\nangiogenesis, increasing PPARγ might be another mechanism that allows tumor cells\nto enhance their survival under these unfavorable conditions (Figure 1).4. IMPACT OF PPARγ AND ITS AGONISTS ON\nANIMAL TUMOR MODELSAnimal models\nwere employed to examine the role of PPARγ in tumors. These systems can be categorized by\nhow the tumor models are generated and by how the dose/activity of PPARγ is altered. With respect to the former, tumors\ncan be generated with xenografts, carcinogens, or genetic manipulations. Watch\nfor spontaneous tumor formation in certain PPARγ genetic backgrounds has also been conducted. With\nrespect to the dose/activity of PPARγ, it can be altered using PPARγ agonists including TZDs or GW7845, or genetic manipulations\nincluding hemizygosity or tissue-specific overexpression or deletion of PPARγ. Results differ drastically between different\nmodel systems, even for the same types of cancer (Tables 5 and 6). This\nreview focuses on models that are more relevant to human cancers. As such, animal\nstudies involving TZD treatment of xenografted tumors are not discussed here.4.1. Colon cancer\nApc+/Min mice possess a\nnonsense mutation in one copy of the adenomatous polyposis coli (APC) gene\nwhich truncates the protein at amino acid 850. Loss-of-function mutations in\nthe APC gene are common in human familial adenomatous polyposis and can\nbe found in sporadic colon cancers as well. Using this model, which is highly relevant\nto human colon cancers, one study showed an increase in tumor number and size, as\nwell as worse histological grade in mice treated with troglitazone or\nrosiglitazone. This is associated with a rosiglitazone-induced increase in the β-catenin protein level in the colon tissues [55]. Another study [56], which also used Apc+/Min mice, reported an\nincrease in the number of colon polyps in troglitazone-treated mice, but\nreported no significant difference in tumor size or histology, which may be\nrelated to the shorter TZD treatments used in this study (5 weeks as compared\nto 8 weeks in the first study). Similar findings were made in Apc\n+/1638N:Mlh1\n+/−\ndouble mutant mice. In these mice, one copy of the APC gene is\ntruncated at amino acid position 1638 and one of the two alleles of the DNA\nrepair enzyme Mlh1 is absent. In the double mutant mice, troglitazone treatment\nsignificantly increased the number of mice that developed large intestine tumors\n[58]. In contrast to these reports, another\nstudy used Apc\n+/1638N mice crossed with hemizygous PPARγ mice.\nBecause homozygous deletion of PPARγ is\nembryonic-lethal, studies examining the dose effect of the gene employed either\na hemizygous Pparγ+/− \nmouse strain or a conditional knock-out strategy. No differences in\nsurvival, number of colonic tumors or β-catenin expression levels were observed\nbetween mice of Apc\n+/1638N :Ppar\nγ\n+/−\nand Apc\n+/1638N :Ppar\nγ\n+/+\nlittermates [57]. Therefore, in colon cancer induced by \nAPC mutations, it appears that activation of PPARγ by TZDs\npromotes tumor formation, while reduction of PPARγ gene\ndosage has little effect on tumor formation.In stark contrast to the APC genetic\ntumor models, carcinogen-generated colon cancer models seem to yield opposite\nresults. In the study that evaluated PPARγ haploinsufficiency\nin an Apc\n+/1638N\nbackground, the investigators also\ndetermined the effect of Pparγ+/− in azoxymethane-mediated colon cancer. Compared to the Pparγ+/+ mice, a greater number of haploinsufficient mice developed tumors in\nthe colon. The tumor-bearing Pparγ+/− mice also had a greater number of tumors in them that led to\nsignificantly decreased survival. In another study, mice with azoxymethane-mediated\ncolon cancer were treated with troglitazone, pioglitazone, or rosiglitazone.\nThis resulted in reduced incidence, number, and size of colorectal tumor [59]. Taken together, these data suggest that PPARγ suppress\nazoxymethane-induced colon carcinogenesis.What would happen in normal mice? Spontaneous\ncolon tumor development was evaluated in normal mice administered with\ntroglitazone [58]. All nine mice fed with troglitazone\ndeveloped tumors in the large intestine, in contrast to none of the 10 mice in\nthe control group. An earlier study did not find any tumors in 17\ntroglitazone-fed normal mice, possibly due to the short duration of feeding (5\nweeks in [56] versus 6 months in [58]).4.2. Mammary gland tumorsThe mammary gland tumor is another\nrelatively well-studied tumor in animals. Similar to colon carcinogenesis, data\non PPARγ's\nrole in mammary gland carcinogensis suggest a wide range of effect depending on\nthe tumor models (Tables 5 and 6). Some studies indicate no effect, while\nothers suggest that it has a tumor promoting role, while others yet suggest a tumor\nsuppressing role. A murine genetic model supports a tumor-promoting role [60]. In this model, the mammary gland tumor is\ninduced by mammary gland-specific expression of polyoma middle T antigen\n(MMTV-PyV). Mammary gland specific constitutive expression of PPARγ\n(MMTV-VpPPARγ) did\nnot yield tumor development. However, when crossed with the MMTV-PyV mice, the\ndouble mutant progeny developed more mammary gland tumors sooner than MMTV-PyV\nmice. The increased tumor burden eventually led to shorter survival.\nInterestingly, hemizygosity of PPARγ in\nthe MMTV-PyV background did not change the time course of tumor development. Exacerbation\nof tumor formation by PPARγ was\nascribed to increased Wnt-β catenin signaling as demonstrated by zebrafish\ndevelopmental models.In contrast to this genetic model,\nchemically induced mammary gland tumors were inhibited by PPARγ agonists.\nBoth TZDs and GW7845, a tyrosine analog, have been shown to exhibit antitumor\neffects. An early study using nitrosomethylurea (MNU) to induce mammary\ncarcinogenesis showed that GW7845 reduced the incidence, number of tumors \nper animal,\nand average weight of tumor at autopsy following a two-month administration of\nthe drug to rats [61]. In 7,12-dimethylbenzanthracene\n(DMBA)-mediated mouse carcinogenesis model, the animals develop multiple types\nof tumor, including mammary ductal papilloma and adenocarcinoma. Incidence of\nmammary gland tumor was significantly higher in Pparγ+/− mice than in Pparγ+/+ mice. The hemizygous mice\nalso had increased number of tumors and a lower survival rate [62].Spontaneous\ntumor formation was also examined in Pparγ+/− mice. Dose reduction of PPARγ does not make animals prone to increased\ncarcinogenesis [62]. In concordance with this\nfinding, the specific deletion of PPARγ in mouse mammary epithelia failed to induce\nmammary tumors in 20 mice observed for 12 months [63].4.3. Other cancersIn a murine prostate\ncancer model, generated using tissue-specific SV40 T antigen, reduced Pparγ+/− had no effects on tumor\nincidence, latency, size, histopathology, or disease progression [64]. However, in a murine follicular\nthyroid cancer model containing a dominant-negative mutant form of thyroid\nhormone receptor β (TRβPV/PV), loss of one PPARγ allele led to increased weight of\ntumor-bearing thyroid gland, increased lung metastasis, and shortened survival.\nIn addition, rosiglitazone treatment of TRβPV/PV mice reduced\nthyroid weight, and tumor progression [65], suggesting a\ntumor-suppressing role for PPARγ. Lastly, in gastric carcinoma, induced with\nMNU, PPARγ haploinsufficient mice had increased tumor\nincidence and shorter survival. Troglitazone treatment significantly reduced\ntumor incidence in mice with wild-type PPARγ background [66].In summary, results\nfrom animal studies regarding the role of PPARγ are conflicting and difficult to assess. For\nthe purpose of clarification, we attempted to analyze the published data\naccording to the cancer types, tumor induction models, PPARγ activation/reduction methods, and tumor\ncharacteristics (Tables 5 and 6). Our extensive analysis revealed no\nclear pattern. However, some trends have been noted: (1) in multiple types of carcinogen-induced tumor (Table 5, light grey shaded rows), PPARγ seems\nto have a tumor-suppressing function. This appears to be independent of how PPARγ is\nactivated or reduced, whereas in genetic tumor models (Table 5, un-shaded rows), the receptor exhibited all possible different effects. As to spontaneous\ntumors (Table 5, dark grey shaded rows), long-term use of troglitazone\nincreased tumor formation, whereas PPARγ reduction\nhad no effect; (2) a reduction of PPARγ dose\nby itself (Table 6, light grey shaded rows) is insufficient to induce spontaneous\ntumor formation, but in existing tumors, it either exacerbates tumor formation\nor have no effect at all; (3)\nTZDs (Table 6, un-shaded rows), in most cases, inhibits tumor\nformation with a rare exception of Apc+/Min mice.The activity of the Wnt/β-catenin signaling pathway might account for\nthese seemingly discrepant results, as tumor models generated by APC mutation\nor polyoma middle T antigen all involve overly active Wnt/β-catenin signaling. TZDs are shown to induce β-catenin in colon [55]. Paradoxically, reduction of PPARγ (Pparγ\n+/−) also increases β-catenin expression in colon [57]. The appropriate activation of PPARγ signaling\nmight also be important. Ligand-independent constitutive activation of PPARγ is\ninvolved in the development of mammary gland tumors [60] as well as in the action of PAX8-PPARγ in\nfollicular thyroid carcinoma [29].5. CLINICAL TRIALS OF TZDs IN\nHUMAN MALIGNANCIESAs discussed above, TZDs have been shown in many\npreclinical studies to possess antitumor effects that have prompted several\nearly-phase clinical studies to evaluate their efficacies in various types of\ncancers. In this review, we analyze these studies both in terms of clinical\nresponses and biological responses, focusing on recently published studies that\ninclude more than 10 patients (Table 7). A phase II clinical trial of rosiglitazone in 12\npatients with liposarcoma was recently conducted. Eight of 12 patients were\nfully evaluated for up to 16 months. As to clinical response, all patients\nprogressed while on treatment with a mean time-to-progression of 5.5 months.\nHistological appearance of repeated biopsy materials did not show any signs of\ntumor differentiation. In one of the 8 patients, PPARγ and fatty acid binding protein (FABP) were\ninduced after 12-week rosiglitazone therapy, but disease in this patient\nprogressed similarly to the others [68]. Ten patients with thyroid\ncancers were treated with rosiglitazone. Among them, 4 had partial response, 2\nhad stable disease, and the remaining 4 progressed. No correlation was found\nbetween the clinical response and levels of PPARγ mRNA and protein in these patients. PAX8-PPARγ status was not assessed [69]. An early study evaluated\nefficacy of troglitazone in 25 patients with metastatic colorectal carcinoma.\nAll 25 patients progressed with a median time-to-progression of 1.6 months and\na median survival time of 3.9 months [70].In breast cancer, data from two human trials have\nbeen published. An early trial on 22 women with refractory breast cancer showed\nno objective response to troglitazone in 18 of the 21 evaluable patients at 8\nweeks after treatment. The therapy was terminated in 16 patients due to\nprogression of their tumors. At 8 weeks, only three patients had stable\ndisease. All patients were evaluated for serum tumor markers, CEA and CA27.29,\nwhich showed increased levels within 8 weeks of treatment. Expression of PPARγ was not determined in the study [71]. A short-term pilot trial of\nrosiglitazone in 38 women with early stage breast cancer was conducted.\nClinical response was not assessed in this short-term (<6 week) study.\nBiological response, as assessed by Ki-67 staining on biopsy tissues before and\nafter treatment, was not detected in treated patients, either. Decreased\ninsulin levels and increased insulin sensitivity were noted in these patients,\nsuggesting that the rosiglitazone did affect metabolism as expected [72].An early phase II trial of troglitazone in 41\npatients with metastatic prostate cancer showed a decrease in levels of\nprostate-specific antigen (PSA) in 20% of patients enrolled in the study.\nProlonged stabilization of PSA was seen in 39% of patients [73]. However, these encouraging\nresults were not reproduced in a large double-blind, randomized, placebo-controlled\ntrial of rosiglitazone in 106 patients with recurrent prostate cancer [74]. The time-to-disease-progression\nwas not significantly different between the rosiglitazone and placebo groups.\nMoreover, the PSA doubling time, a predictor of clinical recurrence, was also\nnot prolonged by the treatment. Taken together, TZDs\nappear to show little benefit, both in terms of clinical response and\nbiological response, in treating various types of human cancers despite\npromising results from preclinical animal studies. It is worth noting that most\nof the studies use low doses of TZDs which are sufficient to activate PPARγ and control diabetes. It remains possible that\nhigher doses, even via receptor-independent pathways, would be beneficial for\ncancer patients. However, one should keep in mind that TZDs are not a class of\ndrugs without dose-limiting toxicities. Troglitazone was withdrawn from the\nmarket by the FDA in 2002 due to liver toxicity. Most recently, increased\ncardiovascular risk has been associated with rosiglitazone in the diabetic\npatient population [75, 76] which has prompted the FDA to\nissue label warnings.6. TZDs AS CHEMOPREVENTIVE AGENTS IN\nEPIDEMIOLOGY STUDIESThe clinical\ntrials discussed above suggest that TZDs have questionable efficacy as\nchemotherapeutic agents in patients who already have cancers. Do they have the\npotential to act as chemopreventive agents? Recently, a large epidemiologic\nstudy, involving a population of 87,678 veteran men with diabetes, attempted to\nanswer that question [77]. In this retrospective study,\nincidence of lung, prostate, and colon cancer in TZD users was compared to\nincidence in non-TZD users and risk of cancer development was analyzed. Only\npatients who obtained a cancer diagnosis after the date of TZD initiation were\nincluded. TZD usage significantly reduced risk of lung cancer by 33%. It also\nreduced risk of colon and prostate cancer, though without statistical\nsignificance. Interestingly, although the risk of prostate cancer is not significantly\ninfluenced by TZDs in the entire population, when examining distinct populations,\nTZDs are associated with an increased incidence of prostate cancer in both Caucasians\nand African Americans. These data suggest that the overall reduced risk is accounted\nfor by the non-Caucasian, non-African Americans populations in the study. These\ndata suggest that TZDs may be beneficial for reducing certain cancers in\ncertain populations. Specific molecular abnormalities in specific cancers and\nthe genetic background of different populations may account for these apparently\ndifferent results.Although this\nstudy was quite strong, we suggest the following for future investigations: (1) separate\nTZD-users into those using rosiglitazone and those using pioglitazone. In the\ncardiovascular risk studies, it was shown that rosiglitazone increases the risk\nwhile pioglitazone decreases the risk [78]. (2) Evaluate the impact of\nthe duration of TZD exposure on risk of cancer development. (3) Determine the\ninfluence of TZDs on the behavior of existing cancers.7. CONCLUSIONSIn this article,\nwe reviewed literature on the roles of PPARγ in cancer with an emphasis on those that\nsuggest a proneoplastic function for the receptor. PPARγ, unlike MYC, RAS, or p53, is neither a strong\ntumor promoter nor a tumor suppressor. However, it may function as a\n“conditional tumor promoter” or a “conditional tumor suppressor” that modulates\nthe tumorigenic process depending upon cellular conditions, tumor types, or\ngenetic background of an animal strain or human individuals. TZDs, as a class\nof pharmacological agent, may have receptor-independent antineoplastic effects,\nespecially at doses higher than diabetic doses or after long-term use and\naccumulation. It remains possible that their antitumor activities would be\nenhanced when in combination with other drugs. Further investigation is needed\nto address that possibility. To help clarify the roles of PPARγ in cancer, future large epidemiological studies\nof diabetic populations with concurrent cancers would be helpful. In addition,\ninvestigations relating PPARγ activities to the clinical outcomes of cancer\npatients would also be informative.\n\nREFERENCES:\n1. LehmannJMMooreLBSmith-OliverTAWilkisonWOWillsonTMKliewerSAAn antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor γ (PPARγ)Journal of Biological Chemistry19952702212953129567768881\n2. RossiAKapahiPNatoliGAnti-inflammatory cyclopentenone prostaglandins are direct inhibitors of IκB kinaseNature2000403676510310810638762\n3. 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BursteinHJDemetriGDMuellerESarrafPSpiegelmanBMWinerEPUse of the peroxisome proliferator-activated receptor (PPAR) γ ligand troglitazone as treatment for refractory breast cancer: a phase II studyBreast Cancer Research and Treatment200379339139712846423\n72. YeeLDWilliamsNWenPPilot study of rosiglitazone therapy in women with breast cancer: effects of short-term therapy on tumor tissue and serum markersClinical Cancer Research200713124625217200362\n73. MuellerESmithMSarrafPEffects of ligand activation of peroxisome proliferator-activated receptor γ in human prostate cancerProceedings of the National Academy of Sciences of the United States of America20009720109901099510984506\n74. SmithMRManolaJKaufmanDSRosiglitazone versus placebo for men with prostate carcinoma and a rising serum prostate-specific antigen level after radical prostatectomy and/or radiation therapyCancer200410171569157415468186\n75. NissenSEWolskiKEffect of rosiglitazone on the risk of myocardial infarction and death from cardiovascular causesThe New England Journal of Medicine2007356242457247117517853\n76. HomePDPocockSJBeck-NielsenHRosiglitazone evaluated for cardiovascular outcomes—an interim analysisThe New England Journal of Medicine20073571283817551159\n77. GovindarajanRRatnasingheLSimmonsDLThiazolidinediones and the risk of lung, prostate, and colon cancer in patients with diabetesJournal of Clinical Oncology200725121476148117442990\n78. RosenCJThe rosiglitazone story—lessons from an FDA advisory committee meetingThe New England Journal of Medicine2007357984484617687124"
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+    "id": "PMC2532707",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2532707\nAUTHORS: Ann V. Schwartz\n\nABSTRACT:\nOver the past two years, evidence has emerged that the currently available thiazolidinediones (TZDs), rosiglitazone, and pioglitazone have negative skeletal consequences, at least in women, which are clinically important. Increased fracture risk in women, but not men, was reported for both TZDs, based on analyses of adverse event reports from clinical trials. In short-term clinical trials in women, both TZDs caused more rapid bone loss. In these trials, changes in bone turnover markers suggest a pattern of reduced bone formation without a change in resorption. Although limited, these results support the hypothesis based on rodent and in vitro models that reduced bone formation resulting from activation of peroxisome proliferator-activated receptor-γ (PPARγ) is a central mechanism for TZDs' effect on bone. Research is needed to better understand the mechanisms of bone loss with TZDs, to identify factors that influence susceptibility to TZD-induced osteoporosis, and to test treatments for its prevention.\n\nBODY:\n1. INTRODUCTIONRecent\nreports have substantially advanced our knowledge of the clinical effects of\nTZDs on skeletal health. In early 2006, research\ninto the skeletal effects in humans of rosiglitazone and pioglitazone, the currently\nprescribed TZDs, was limited to observational studies [1]. Although a body of evidence had developed\nfrom rodent and in vitro studies that these two TZDs cause bone loss, it was\nnot known if these compounds had a similar effect in humans. Since then, rosiglitazone and piogltiazone were\neach linked to increased fracture risk among diabetic women, based on adverse\nevent reports in clinical trials. And,\nin women, short-term clinical trials demonstrated substantial bone loss with\nboth TZDs. Pioglitazone and\nrosiglitazone are widely used to treat diabetes, and better knowledge of their\nskeletal effects is crucial to guide clinical decisions. At the same time, because TZDs are ligands of\nPPARγ, a better understanding of their skeletal effects will help to clarify\nthe role of PPARγ in bone metabolism and potentially shed light on the\nmechanisms of age-related bone loss. This\nreview considers the recent clinical evidence regarding TZDs and skeletal\nhealth and discusses outstanding issues that warrant further research.2. ROSIGLITAZONE AND FRACTURE RISKEvidence\nthat RSG increases fracture risk emerged with the results of the ADOPT trial\npublished in 2006 [2]. A postproof note in the main report from the\ntrial indicated increased fracture risk in women, but not men, enrolled in the\ntrial. Since then, the fracture results\nhave been published separately and in more detail [3]. ADOPT was designed to assess time to\nmonotherapy failure for RSG compared to metformin and to a sulfonylurea,\nglyburide. The trial had three arms,\ncorresponding to the three different treatments, and enrolled a total of 2511\nmen and 1840 women who were followed for a median of 4.0 years. The average age was 57 years. By\nself-report, 77% of women were postmenopausal. \nParticipants were recently diagnosed with diabetes (<3 years), were\ndrug naïve for hypoglycemic medications, and had an average A1C of about\n7.4%.Fractures,\nidentified through adverse event reports, were specifically reviewed after the\nconclusion of the trial. Based on time\nto first fracture, the investigators found an increased risk among women in the\nRSG arm of 1.81 (95% CI: 1.17, 2.80) compared to metformin, and 2.13 (1.30,\n3.51) compared to glyburide. The risk\nfor men was not increased compared with either metformin (RH 1.18; 95% CI:\n0.72, 1.96) or glyburide (RH 1.08: 95% CI: 0.65, 1.79).In women,\nrisk was increased for both upper and lower limb fractures. Rate ratios calculated from\nfracture rates\nreported for ADOPT showed the largest increases in relative risk for foot (RR = 3.3),\nhand (RR = 2.6), and proximal humerus (RR > 8) fractures (see Table 1). There was no increased risk identified for\nclinical spine or hip fractures, but the numbers of these fractures, 3 clinical\nspine and 4 hip fractures among all women, were too small to draw firm\nconclusions. The small number of hip\nand spine fractures in the ADOPT population (average age 57 years) is not\nsurprising since the rate of these fractures tends to be relatively low until\nafter age 65.For\nwomen, an examination of the survival curves from the ADOPT trial (see Figure 1)\nsuggests that the increased risk of fracture with RSG is evident after about\none year of treatment. In separate\ntrials, discussed below, bone loss could be identified among women treated with\nRSG after only a few months of treatment. However, the ADOPT results suggest that bone\nloss with RSG does not make a noticeable difference in fracture risk until after\nabout 12 months of treatment.Self-reported\nmenopausal status and baseline use of estrogen-containing hormones were available\nfor women enrolled in ADOPT. As\nexpected, premenopausal women had a lower rate of fracture than postmenopausal\nwomen, but both groups had an approximate doubling of fracture risk with RSG\ntreatment. Menopausal status did not\nappear to substantially modify the effects of RSG on fracture. About 20% of women reported use of an\nestrogen-containing hormone at baseline. \nThe effect of RSG on fracture risk did not appear to differ between\nthose who did or did not report estrogen use.It is\npossible, though not established, that poor glycemic control increases fracture\nrisk [6]. However, this would not explain the ADOPT\nresults as those in the RSG arm maintained glycemic control on monotherapy\nlonger than those in the metformin or glyburide arms.3. PIOGLITAZONE AND FRACTURE RISKWith\nthe published report of increased fracture risk in the RSG arm of ADOPT, Takeda Pharmaceuticals, IL, USA the manufacturer of\npioglitazone, reviewed their clinical trial databases and, in a letter to\nhealth care providers in 2007, reported an increased fracture risk with\npioglitazone treatment in women, but not men [7]. The databases included 24 000 years of\nfollowup for over 8100 patients treated with pioglitazone and over 7400\npatients in the comparison group. In\nthese trials, the maximum duration of pioglitazone use was only 3.5 years. The magnitude of the increased risk reported\nfor all clinical fractures was similar to the ADOPT results with a fracture\nrate of 1.9 per 100 person years in those using pioglitazone compared with a\nrate of 1.1 per 100 person years in those using placebo or an active comparator\ndrug. The relative risk for men was not\nreported but was stated to be not statistically significant. Data on specific fracture sites was not\nprovided although the letter stated that most of the fractures occurred in the\ndistal upper limb or distal lower limb.4. TZDs AND BONE LOSSIn 2007,\nGrey et al. reported the results of a 14-week randomized clinical trial\ncomparing RSG (8 mg/day) with placebo in 50 postmenopausal women, average age 67\nyears, who did not have diabetes or osteoporosis [8]. The trial found modest reductions in two\nmarkers of bone formation. Procollagen\ntype-I N-terminal propeptide was reduced by 13% (P = .004) and\nosteocalcin by 10% (P = .04) in the RSG arm compared with placebo. In contrast, the bone resorption marker, serum\nβ-C-terminal telopeptide (S-CTX) of type I collagen, was stable in the RSG arm and\ndid not differ significantly from placebo (P = .9). Substantial bone loss was reported at the\ntotal hip with RSG treatment. Women in\nthe RSG group lost bone density (BMD) more rapidly at the total hip (−1.9% RSG\nversus −0.2% placebo, P = .003). For the total spine, bone loss was more rapid in the RSG arm but the\ndifference was not statistically significant (−1.2% RSG versus −0.2% placebo, P = .13).In a\nrandomized, controlled, but unblinded trial, a lower dose of RSG (4 mg/day) for\n12 weeks was compared with diet treatment alone in obese postmenopausal women\nwith newly diagnosed diabetes [9]. Bone-specific alkaline phosphatase, a bone\nformation marker, was decreased in the RSG arm (−21.5%) compared with diet only\n(−4.1%) (P < .05). Osteocalcin\nwas decreased similarly in both arms (RSG −20%; diet only −17.6%) while urine\ndeoxypyridinoline (DPD), a resorption marker, was not increased in the RSG arm\n(3%) compared with the diet only arm (17%).The\nshort-term effects of pioglitazone (30 mg/day) on bone density and markers have\nbeen tested in a 16-week randomized placebo-controlled trial among 30\npremenopausal women with polycystic ovary syndrome (PCOS) [10]. BMD was reduced compared with placebo at the\nlumbar spine (−1.14% versus 0.00%), total hip (−0.18% versus 1.35%), and\nfemoral neck (−1.45% versus 0.87%) (all P < .05). The magnitude of loss in the PIO group at the\nspine and femoral neck is similar to BMD losses reported with RSG use over 14\nweeks in postmenopausal women [8]. Alkaline phosphatase, a marker of bone\nformation, was decreased in the PIO group compared to placebo but osteocalcin\nwas not. Changes in the marker of bone\nresorption, S-CTX, were also not significantly different across treatment\ngroups. The treated group experienced a significant\ndecrease in fasting insulin compared to placebo. Since insulin may be anabolic for bone, this\nmay have contributed to the bone loss observed with PIO although the authors reported\nthat the changes in BMD and the changes in insulin were not significantly\ncorrelated. Estradiol and testosterone\nlevels were not significantly altered in the PIO group.Two\nobservational studies have reported results for TZDs and changes in BMD or\nmarkers. The first clinical study to\nreport increased bone loss with TZD use, combining troglitazone, rosiglitazone,\nand pioglitazone, was based on the Health, Aging, and Body Composition\nlongitudinal observational study of older adults [11]. The cohort included 666 diabetic participants\nwith an average age of 73 years. Of\nthese, 69 participants reported any TZD use during four years of followup. Increased bone loss was found in diabetic\nwomen but not men. After controlling for\npotential confounders, the additional bone loss attributed to TZD use in women was\n−1.23% (95% CI: −2.06%, −0.40%) per year at the lumbar spine, −0.61% (−1.02%,\n−0.21%) per year for whole body, and −0.49% (−1.04%, 0.07%) for total hip. These estimates of increased bone loss are\nsubstantially lower than those reported by Grey et al. [8]\nfor the trial of RSG use and by Glintborg et al. [10]\nfor the trial of PIO use. The additional\nbone loss of 1.5–1.7% at the total hip over 14–16 weeks in these\ntwo trials, if sustained, would result in additional bone loss of 5-6% annually. While the observational study by Schwartz et\nal. may have underestimated the degree of bone loss associated with TZD use, it\nseems unlikely that bone loss of 6% per year is occurring with TZD use. Instead, there may be an initial period of more\nrapid bone loss, followed by continued loss at a lower rate, similar to the\neffect of glucocorticoids [12].Although\nSchwartz et al. reported no increased bone loss with TZD use in diabetic men,\nYaturu et al., in an observational study of 160 older diabetic men (average age\n68 years), did report that RSG use (N = 32) was associated with increased bone\nloss of −1.05% per year at the total hip, −1.02% at the femoral neck, and\n−1.24% at the spine (all P < .03) [13]. However, the study did not have sufficient\npower to control for potential confounders such as A1C level, use of other\nmedications, or diabetic complications.4.1. Rodent\nand in vitro modelsResults\nof rodent and in vitro models provided the first evidence that RSG and PIO\ncause bone loss. RSG has been more extensively\nstudied in these models but both compounds are associated with bone loss in\nrodents [14, 15]. These findings have been reviewed previously\n[16, 17]\nand will not be discussed in depth here. \nHowever, a few points are worth noting as particularly relevant to\nfuture research in humans. In general,\nthese models indicate a negative effect on osteoblast differentiation and\nactivity with a decrease in bone formation. However, in a few reports, TZDs were\nassociated with increased resorption. \nNotably, this occurred in ovariectomized rats [18]\nand in aged mice [19]. Sottile et al. reported that ovariectomized\nrats experienced bone loss with RSG, but intact female rats did not, and that\nthe bone loss was characterized by increased resorption [18]. This suggests an interaction between RSG and\nestrogen levels that needs to be assessed in human studies. The results from Lazarenko et al. comparing\nthe effects of RSG in young, adult, and aged mice suggest that the mechanism of\naction may be different in the aged mice [19]. In young and adult mice, bone loss with RSG\ntreatment was driven by reduced formation while in older mice RSG treatment\nresulted in increased resorption. These\nresults need to be explored in human studies as they would suggest different\napproaches to treatment for the prevention of TZD-induced osteoporosis.5. FUTURE DIRECTIONS FOR CLINICAL RESEARCHSubstantial\nevidence has now emerged that RSG and PIO have clinically important negative\nskeletal effects. Increased fracture\nrisk in women, but not men, has been reported for both RSG and PIO. Although this increased fracture risk was\nidentified in the context of clinical trials, the fractures were identified\nthrough adverse event reports and were not a planned outcome of the\ntrials. It is possible for adverse event\nresults in a clinical trial to give a signal that is statistically significant\ndue to chance rather than to an actual effect of the intervention. However, the fracture effect is consistent\nwith two clinical trials demonstrating bone loss with RSG and PIO. And, the increased fracture risk and bone\nloss are consistent with the results of rodent and in vitro models. The combination of these studies provides a\ncompelling argument that, in women, the two currently prescribed TZDs cause\nhigher fracture risk due to bone loss.Given\nthis growing evidence of increased fracture risk and bone loss with TZD use, further\nexploration of the skeletal effects of TZDs is crucial to inform efforts to\nprevent TZD-induced osteoporosis and, more generally, to delineate the role of\nPPARγ in bone metabolism. Some of the\nkey questions for clinical research are identified and discussed below.5.1. What groups are at higher risk?To\ninform clinical decisions and to better understand the mechanism of TZDs effects\non the skeleton, it is important to ascertain if there are groups that are\nparticularly vulnerable, or groups that are not susceptible, to increased\nfracture risk with TZD use. So far, the\nnegative skeletal effects seem to be more important for women than for men, but\nresults are not conclusive. Among\nwomen, menopausal status does not appear to modify the effect of RSG on the\nskeleton. The ADOPT results indicate\nthat increased fracture risk extends to those who are premenopausal as well as\npostmenopausal. Both premenopausal [10]\nand postmenopausal [8]\nwomen have been shown to lose bone with TZD treatment.A\npossible explanation for the lack of effect on the skeleton in men is the\nhigher estrogen levels found in older men compared with older women. In a rat model, ovariectomized, but not\nintact, females had bone loss with RSG treatment, suggesting a protective\neffect from higher estrogen levels [18]. However, clinical results to date indicate\nthat TZDs cause increased bone loss and fracture risk in pre- as well as\npostmenopausal women. Further research with\nmeasurements of endogenous estrogen levels could clarify whether there is an\ninteraction between estrogen levels and TZD use.5.2. What happens to bone density and turnover after 3-4 months of\ntreatment?The randomized\ntrials with RSG and PIO have reported on treatment for 14–16 weeks. In both trials, the additional bone loss in\nthe treated group was substantial, equivalent to a loss of 5-6% over a year,\nbut it seems unlikely that this rate of loss is being sustained over longer\ntreatment periods. Observational studies\nsuggest increased loss of about 0.5–1% each\nyear. Steroid treatment appears to cause\ninitial rapid bone loss followed by continued loss but at a lower rate; the TZDs may present a similar pattern [12]. However, trials of longer duration are needed\nto assess the degree of loss over several years.5.3. Effect on resorption as well as formation?One\nof the key questions regarding the mechanism of action of the TZDs is whether\nbone resorption and formation, or only one, are affected. The clinical evidence to date, based on bone\nturnover markers, points to a reduction in bone formation without a change in\nbone resorption. However, these results\nare based on only three studies that included bone marker results [8–10]. Rodent models have\ngenerally shown reduced bone formation but, in aged mice and in ovariectomized\nrats, bone resorption is increased. \nWhether bone resorption is similarly increased with older age or with\nvery low endogenous estrogen levels in human studies has not been fully\nexplored.5.4. Do effects on cortical and trabecular bone differ?The\nincreased fracture risk observed in the bones of the extremities, that have a\nrelatively high proportion of cortical bone, suggests a negative impact on\ncortical bone. This pattern is distinct\nfrom glucocorticoids which have a particularly strong effect on trabecular bone\nand the risk of vertebral fracture [12]. Studies using imaging techniques that can separate\nthese two compartments, such as high resolution computed tomography, could\nclarify whether the effects of TZDs differ for cortical and trabecular bones.5.5. Marrow adiposityIn\nmost reports from rodent models, increased marrow adiposity accompanies bone\nloss with RSG treatment. Further\ninvestigation of this phenomenon has suggested that activation of PPARγ with\nRSG increases lineage allocation of stem cells towards adipocytes at the\nexpense of osteoblasts in the marrow. To date, human studies have not measured bone marrow adiposity. Knowledge of the effect of TZDs on bone\nmarrow fat would increase our understanding of the mechanisms underlying bone\nloss and fracture risk in humans with TZD use. \nIn addition, an increase in bone marrow fat may cause an artificial\ndecrease in BMD measured by DXA [20]. If marrow fat is increased, the degree\nof bone loss with TZD use may be overestimated by DXA measurements.5.6. Effective treatment for TZD-induced osteoporosisThere\nare no studies to date on treatments that might prevent TZD-induced bone\nloss. Although the bisphosphonates\nmainly target bone resorption, the general reduction in bone turnover may be\nefficacious in preventing bone loss with TZD treatment. The bisphosphonates are successfully used for\nprevention of osteoporosis with corticosteroid treatment, also characterized by\nreduced bone formation [21]. However, TZDs have specific effects on bone,\nand bisphosphonate use should be explicitly tested to determine efficacy in\nthis situation. Treatments\nthat increase bone formation, currently limited to parathyroid hormone (PTH)\nand strontium ralenate, could theoretically prevent TZD-induced bone loss. PTH has been shown to prevent bone loss with\nglucocorticoid therapy [22],\nbut neither treatment has been tested in relation to TZDs.6. CONCLUSIONResearch\nover the past two years has provided new clinical evidence that the currently\nprescribed TZDs increase fracture risk and bone loss, at least in women. Combined with the findings from rodent and in\nvitro models, these clinical results suggest that activation of PPARγ can play\na role in bone loss. With the widespread\nuse of TZDs as a diabetes treatment, further research is needed to delineate\nthe groups that are most susceptible to TZD-induced osteoporosis, to determine\nthe rate of bone loss with TZD treatment beyond 16 weeks, to assess the effects\nof TZDs on marrow adiposity, cortical and trabecular bones, and to identify\ntreatments to prevent TZD-induced fracture risk. Addressing these questions will advance our\nability to prevent TZD-induced osteoporosis and will provide a better\nunderstanding of the role of PPARγ activation in bone metabolism.\n\nREFERENCES:\n1. SchwartzAVDiabetes, TZDs, and bone: a review of the clinical evidencePPAR Research200620066 pagesArticle ID 24502.\n2. KahnSEHaffnerSMHeiseMAGlycemic durability of rosiglitazone, metformin, or glyburide monotherapyThe New England Journal of Medicine2006355232427244317145742\n3. KahnSEZinmanBLachinJMRosiglitazone-associated fractures in type 2 diabetes: an analysis from ADOPTDiabetes Care200831584585118223031\n4. GlaxoSmithKline. (GSK)Clinical trial observation of an increased incidence of fractures in female patients who received long-term treatment with Avandia® (rosiglitazone maleate) tablets for type 2 diabetes mellitus (Letter to Health Care Providers), February 2007, http://www.fda.gov/MedWatch/safety/2007/Avandia_GSK_Ltr.pdf.\n5. SchwartzAVSellmeyerDEEffect of thiazolidinediones on skeletal health in women with Type 2 diabetesExpert Opinion on Drug Safety200871697818171315\n6. SchwartzAVDiabetes mellitus: does it affect bone?Calcified Tissue International200373651551914517715\n7. TakedaObservation of an increased incidence of fractures in female patients who received long-term treatment with ACTOS®\n(pioglitazone HCl) tablets for type 2 diabetes mellitus. (Letter to Health Care Providers), March 2007, http://www.fda.gov/medwatch/safety/2007/Actosmar0807.pdf.\n8. GreyABollandMGambleGThe peroxisome proliferator-activated receptor-γ agonist rosiglitazone decreases bone formation and bone mineral density in healthy postmenopausal women: a randomized, controlled trialThe Journal of Clinical Endocrinology & Metabolism20079241305131017264176\n9. BerberogluZGursoyABayraktarNYaziciACBascil TutuncuNGuvener DemiragNRosiglitazone decreases serum bone-specific alkaline phosphatase activity in postmenopausal diabetic womenThe Journal of Clinical Endocrinology & Metabolism20079293523353017595249\n10. GlintborgDAndersenMHagenCHeickendorffLHermannAPAssociation of pioglitazone treatment with decreased bone mineral density in obese premenopausal patients with polycystic ovary syndrome: a randomized, placebo-controlled trialThe Journal of Clinical Endocrinology & Metabolism20089351696170118285411\n11. SchwartzAVSellmeyerDEVittinghoffEThiazolidinedione use and bone loss in older diabetic adultsThe Journal of Clinical Endocrinology & Metabolism20069193349335416608888\n12. van StaaTPLeufkensHGMCooperCThe epidemiology of corticosteroid-induced osteoporosis: a meta-analysisOsteoporosis International2002131077778712378366\n13. YaturuSBryantBJainSKThiazolidinediones treatment decreases bone mineral density in type 2 diabetic menDiabetes Care20073061574157617363747\n14. RzoncaSOSuvaLJGaddyDMontagueDCLecka-CzernikBBone is a target for the antidiabetic compound rosiglitazoneEndocrinology2004145140140614500573\n15. JennermannCTriantafillouJCowanDPenninkBConnollyKMorrisDEffects of thiazolidinediones on bone turnover in the ratJournal of Bone and Mineral Research199510p. S241(Abstract S361)\n16. Lecka-CzernikBSuvaLJResolving the two “bony” faces of PPAR-γ\nPPAR Research200620069 pagesArticle ID 27489.\n17. GreyASkeletal consequences of thiazolidinedione therapyOsteoporosis International200819212913717901911\n18. SottileVSeuwenKKneisselMEnhanced marrow adipogenesis and bone resorption in estrogen-deprived rats treated with the PPARgamma agonist BRL49653 (rosiglitazone)Calcified Tissue International200475432933715549648\n19. LazarenkoOPRzoncaSOHogueWRSwainFLSuvaLJLecka-CzernikBRosiglitazone induces decreases in bone mass and strength that are reminiscent of aged boneEndocrinology200714862669268017332064\n20. HangartnerTNJohnstonCCInfluence of fat on bone measurements with dual-energy absorptiometryBone and Mineral19909171812337690\n21. ReidDMHughesRALaanRFJMEfficacy and safety of daily risedronate in the treatment of corticosteroid-induced osteoporosis in men and women: a randomized trialJournal of Bone and Mineral Research20001561006101310841169\n22. LaneNESanchezSModinGWGenantHKPieriniEArnaudCDParathyroid hormone treatment can reverse corticosteroid-induced osteoporosis. Results of a randomized controlled clinical trialThe Journal of Clinical Investigation19981028162716339788977"
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+    "id": "PMC2532744",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2532744\nAUTHORS: Philip Barnes, Kerrie L. Thomas\n\nABSTRACT:\nIt is essential to understand the molecular processes underlying long-term memory to provide therapeutic targets of aberrant memory that produce pathological behaviour in humans. Under conditions of recall, fully-consolidated memories can undergo reconsolidation or extinction. These retrieval-mediated memory processes may rely on distinct molecular processes. The cellular mechanisms initiating the signature molecular events are not known. Using infusions of protein synthesis inhibitors, antisense oligonucleotide targeting brain-derived neurotrophic factor (BDNF) mRNA or tPA-STOP (an inhibitor of the proteolysis of BDNF protein) into the hippocampus of the awake rat, we show that acquisition and extinction of contextual fear memory depended on the increased and decreased proteolysis of proBDNF (precursor BDNF) in the hippocampus, respectively. Conditions of retrieval that are known to initiate the reconsolidation of contextual fear memory, a BDNF-independent memory process, were not correlated with altered proBDNF cleavage. Thus, the processing of BDNF was associated with the acquisition of new information and the updating of information about a salient stimulus. Furthermore, the differential requirement for the processing of proBDNF by tPA in distinct memory processes suggest that the molecular events actively engaged to support the storage and/or the successful retrieval of memory depends on the integration of ongoing experience with past learning.\n\nBODY:\nIntroductionTranscriptional and post-translational molecular events are required for the consolidation of information into long-term memories and are thought to lead to the synaptic structural changes that maintain the memory [1], [2]. Originally described by Pavlov (1927), extinction occurs when a conditioned stimulus (CS) is presented without reinforcement of a biologically salient unconditioned stimulus (US), manifesting as a weakening of the conditioned response. Although historically extinction has been viewed as unlearning [3], [4], 5, it is currently viewed as the generation of a new memory about a CS [6]. The extinction memory competes with the original memory for control of behaviour. The protein synthesis-dependent nature of extinction [7] perhaps further emphasises that extinction is a long-lasting memory that is independently acquired and stored.The molecular mechanisms underlying the formation of long-term fear memory [8], [9], [10] share a remarkable similarity with those required for the primary model of memory formation in neuronal circuits, long-term-potentiation [11]. The activation of these particular molecules may contribute to the enhancement of synaptic strength in the hippocampus and amygdala observed upon the encoding of fear memory [12], [13]. Similar plasticity-related molecular processes maybe required for consolidation and extinction [14], [15], [16], [17]. However, reports indicating that activation of CB1, calcineurin and PI3K-dependent signalling pathways are selectively required for the extinction of fear memory [18], [19], [20], not only suggest that the molecular processes of extinction dissociate from those of consolidation but may more closely correlate with the plasticity processes of long-term depression or depotentiation [21], [22], [23].We have recently shown in vivo that the activity of the secreted neurotrophin, brain-derived neurotrophic factor (BDNF) in the hippocampus is required for the consolidation of hippocampal-dependent contextual fear memory [24]. We also showed that reconsolidation, the restabilisation of the labile memory following the recall by a brief exposure to a reminder stimulus, was not dependent on BDNF. More specifically, we showed that consolidation was critically dependent on the mature form of the neurotrophin, mBDNF. mBDNF is generated by the proteolytic cleavage of the precursor, proBDNF, by protease tissue plasminogen activator (tPA)-mediated activation of plasmin [25], [26]. Studies in in vitro preparations have compellingly shown the requirements for mBDNF and proBDNF for hippocampal LTP and LTD respectively [27], [28]. Here using a strategy of independently manipulating two fear memories in the same animal, and using temporally and regionally restricted manipulations of BDNF levels, we show that the processing of proBDNF is positively correlated with the acquisition but negatively correlated with extinction.Materials and MethodsSubjectsThe subjects were adult male Lister hooded rats weighing 280–350 g. They were housed in pairs, in holding rooms maintained at 21°C on a reversed-light cycle (12 h light/dark; lights on at 10:00 P.M.). All experiments were conducted in the dark period of the rats. Food and water were freely available throughout the experiment. All procedures were conducted in accordance with local Cardiff University Ethical Committee approval and the United Kingdom 1986 Animals (Scientific Procedures) Act (Project license PPL 30/2236).Surgery, ODN, ANI and tPA STOP infusions, and histological assessment of cannula placementPerformed as described by [24] with the exception that the rats were anaesthetised using isoflurane [flow rates: -oxygen; 0.8 liter/minute, NO2; 0.4 litre/minute] and were implanted with stainless steel double guide cannulae (Plastics One, 22 gauge, 3.8 mm centre-to centre, 3 mm below pedestal) aimed at the dorsal hippocampus (AP -3.50, relative to bregma). Stainless steel double cannulae 1 mm longer than guide cannulae was inserted into the guide cannulae to maintain patency during recovery. Subsequent histological analysis revealed accurate placements in all cannula-implanted rats (data not shown). Infusions were carried out using a syringe pump, connected to injectors (28 gauge, projecting 1 mm beyond the guide cannulae) by polyethylene tubing. ODNs were PAGE-purified phosphorothioate end-capped 18-mer sequences, resuspended in sterile PBS to a concentration of 1 nmol/µl: BDNF antisense ODN, ASO, 5′-TCT TCC CCT TTT AAT GGT-3′; BDNF missense ODN, MSO, 5′-ATA CTT TCT GTT CTT GCC-3′. All ODN sequences were subjected to a BLAST search on the National Center for Biotechnology Information BLAST server using the Genbank database. Antisense sequences had positive matches only for their target mRNA sequences, and no other rat or human coding sequences. Control missense sequences, which included the same 18 nucleotides as the ASO but in a scrambled order, did not generate any full matches to identified gene sequences in the database. Anisomycin (ANI, 80 mg/ml, Sigma) and tPA-STOP (4 mM, ADI) was dissolved in sterile PBS. The PBS vehicle was used for habituation infusions in all rats 1 day before conditioning and to act as control infusions on the day(s) of training. ODNs (1.0 µl per side; 0.125 µl/min) or tPA-STOP (1.5 µl per side; 0.125 µl/min) were infused 90 min prior to conditioning or context reexposure, and ANI (1.0 µl per side; 0.5 µl/min) were infused immediately after context reexposure.On completion of the behavioural testing, the rats were killed by CO2 asphyxiation, and the brains removed and fixed in 4% fresh paraformaldehyde in 0.1 M phosphate buffered saline for at least 48 hours before being transferred to 20% sucrose in PBS solution for cryoprotection. Forty-micrometer coronal sections through the dorsal hippocampus were cut on a freezing microtome, mounted onto gelatine-coated slides and Nissl-stained with thionin. The sections were examined under a light microscope and the subjects were only included if the infusion cannulae tracts terminated bilaterally in the hippocampus and there was no damage to adjacent brain structure or gross ventricular enlargement. Subsequent histological analysis revealed accurate placements in all cannula-implanted rats (data not shown).SDS-PAGE and Western BlottingFollowing fear conditioning/retrieval test rats were sacrificed by carbon dioxide inhalation. The rats were decapitated and the brain was rapidly removed and placed on ice. The hippocampal dentate gyrus/CA3 and CA1 regions were microdissected and frozen on dry ice prior to storage at −80°C. Tissue lysates and Western blotting were performed essentially as previously described [24]. Proteins (4–10 µg) were separated on 16.5% Tris-Tricine gels at a constant voltage of 80 V and then transferred to Hybond-P PDVP membranes (Amersham Biosciences) at a constant voltage of 100 V for 1 hr. Blots were blocked in 5% non-fat in 0.01 M Tris-buffered saline solution containing 1% Tween 20 (TBST), and this TBST solution was used for all subsequent washes. Primary and secondary antibodies were diluted in TBST containing 0.5% Tween 20 and were used at the following concentrations: Arc (H-300 Santa Cruz), 1∶10000; BDNF (AP1779SP, Chemicon), 0.1 µg/ml, β-actin (AbCam), 1∶20 000; goat anti-rabbit and goat anti-mouse IgG (whole-molecule)-peroxidase conjugates (Sigma), 1∶10 000). Blots were developed using ECL Advance detection (Amersham Biosciences) and opposed to autoradiographic film. Autoradiographs of each Westerns blot were developed to be linear in the range used for densitometry for each protein target and for β-actin. Autoradigraphic images were captured on a Sharp JX330 Scanner using Labscan v2.0 (Pharmacia Biotech) and quantified using ImageMaster 1D Prime v.3.0 (Amersham Pharmacia Biotech). For analysis, optical density (OD) values and the band areas were obtained for each microdissected hippocampal sample for both the target protein (Arc/Arg3.1, BDNF) and the β-actin loading control to derive an amount figure. Averaging the amount of β-actin across samples on each Western blot and deriving a normalization factor for each sample corrected loading variation.Contextual Fear Conditioning in Two ContextsEach rat received two conditioning trials in two different contexts separated by 24 hours. Individually, rats were first pre-exposed for 3 d to two experimental chambers (contexts) for 20 min/d. These contexts were designed to differ in a number of features including size, spatial location, odor, and lighting. In addition, to further distinguish the two contexts, exposure to each chamber was separated by a minimum of 4 hours. The first conditioning trial was given 24 hours later. Conditioning consisted of the rats being placed individually in a chamber for 3 min. After 2 min a single scrambled footshock (0.5 mA for 2 s) was delivered. After 24 hours the rats were returned to the other conditioning chamber for 3 min and they received a single scrambled footshock (0.5 mA for 2 s) after 2 min. The order of the contexts that the rats were conditioned to was counterbalanced in each experiment. Extinction training: Each rat received two extinction training trails in the two different conditioned contexts separated by 24 hours. One or two days after contextual fear conditioning, rats were re-exposed to one of the conditioned contexts either for 2 or 10 min. 24 hours later the rats were exposed to the other conditioned training contexts for either 2 min or 10 min. The order of the conditioned contexts that the rats were exposed to during extinction training was counterbalanced. Retrieval tests: Four or five days, and sometimes 14 days, later each rat was given two contextual fear memory retrieval tests (T1 and T2, respectively) separated by 24 hours. The rats were placed into one of the conditioned contexts for 2 min and the following day they were exposed for 2 min to the other conditioned context. The order to which each rat was exposed to the two contexts during the retrieval trails was the same as during the conditioning training.Contextual Fear Conditioning in a Single ContextWhere indicated, rats were habituated to handling by placing them for 20 min in one of two distinct conditioning contexts for 3 d (for details see above), the final habituation session preceding conditioning by 24 hrs. During a 3 min conditioning training trial, rats received a single scrambled footshock (0.5 mA for 2 s) 2 min after being placed into the conditioning context. Extinction training 3 d later consisted of exposing rats to the conditioned context for either 2 min or 10 min.Analysis and StatisticsFreezing behavior served as a measure of conditioned fear to the contexts during the conditioning, extinction training and retrieval tests of the behavioural procedures. This was video-recorded and quantified by an observer blind to the experimental group. One unit of freezing was defined as a continuous absence of movement other than that required for respiration in 1 s sampled every 10 s. Data are presented as the Mean±S.E.M. percentage time spent freezing. Freezing behaviour was analyzed in a repeated measures analysis of variance (repeated measures ANOVA) with test as a within-subjects factor or by ANOVA. For repeated measures ANOVA, Mauchly's Test of Sphericity was applied. If the sphericity assumption was not met, the Greenhouse-Geisser correction was applied. Post-hoc planned comparisons were made using repeated measures ANOVA and the P value constrained by the number of comparisons made. ANOVA was applied to data from Western blot experiments. Tukey's test was then used for post hoc analysis to determine the sources of significance (P<0.05, P<0.01 and P<0.001).ResultsContextual fear memories can be independently manipulated by context exposureA powerful method of measuring the effects of experimental manipulations on memory stability after recall would be to show that the manipulation selectively impacted on a recalled memory but would leave a non-recalled memory intact. Therefore, we first established a behavioural protocol by which two different hippocampal–dependent contextual fear memories (CFM) could be separately retrieved and manipulated by the duration of the exposure to the conditioned context during extinction training (Fig. 1). Firstly, rats were fear conditioned to two different contexts (A and B) by presenting a short unsignalled footshock in each of the contexts on consecutive occasions. During extinction training each rat simply received a 2 min exposure to one of the conditioned contexts and a 10 min exposure to the other conditioned context. The exposures to the contexts during the behavioural training sessions were counterbalanced across the experiment. The effect of extinction training on conditioned freezing behaviour (an index of fear memory) was also measured during two series of context re-exposure recall tests 5 and 14 days later. This protocol is illustrated in Fig. 1. Rats showed a robust conditioned freezing behaviour in the two contexts during the first 2 min of each extinction training session indicating CFM had been established for both contexts. During the recall test 5 days later (LTM1), rats characteristically showed less freezing in the context in which they had received a 10 min exposure during extinction training (A) than in the context they were exposed to for 2 min during extinction training (B, Fig. S1). We showed in a similar contextual fear conditioning procedure that a 2 min exposure to a conditioned context engaged reconsolidation processes which stabilise or maintain the fear memory for subsequent recall, as measured by high levels of conditioned freezing at all recall tests [24]. Here likewise, the maintenance of high levels of freezing in context B at LTM1 suggest reconsolidation of the fear memory for context B was induced by brief exposure to this particular context during extinction training. In the same animals, a longer 10 min exposure to context A at extinction training induced the extinction of fear memory for context A. Thus, two separate CFM could be independently modified by their context-selective recall and the conditions (duration of context re-exposure) of recall. There was no recovery of the extinguished fear memory at the second recall test, LTM2, 19 days after extinction training.10.1371/journal.pone.0003248.g001Figure 110 min exposure to a conditioned context induced the extinction of a selectively recalled fear memory.Repeated measures ANOVA revealed significant effects of the training and test phases on freezing behaviour (F (4.329, 47.618) = 11.355, P = 0.000, ε = 0.481). Rats (n = 12) presented with a single footshock (US) in two distinct contexts (A and B) 24 hrs apart (C1 and C2) showed robust freezing behaviour in the post US period and during the first 2 min of exposure to the conditioned contexts during the two extinction training sessions (E1 and E2) two days later. All rats experienced a 2 min re-exposure to one of the conditioned contexts and a 10 min exposure to the other conditioned context in a counterbalanced manner. Rats re-exposed to the conditioned contexts 5 days later (LTM 1) showed less freezing in the context in which they experienced a 10 min exposure (A), than in the context that they had been exposed to for 2 min (B) during extinction training. At a further test 3 weeks after conditioning (LTM 2) the rats showed low levels of conditioned fear in both contexts. Results are presented as the Mean±S.E.M. * P<0.05, ** P<0.01, *** P<0.001.The extinction of contextual fear memory is dependent on protein synthesis in the hippocampusTo determine whether the extinction of contextual fear memory was dependent on the hippocampus, and more specifically required protein synthesis in this brain region, we used a similar behavioural training procedure to the previous experiment except that during extinction training each rat received a 10 min exposure to both of the conditioned contexts. The protein synthesis inhibitor, ANI, and PBS were infused into the hippocampus immediately after extinction training sessions E1 and E2 (Fig. 2). Infusions were administered in a counterbalanced fashion such that half the rats received ANI at E1 and PBS at E2, and vice versa for the remaining rats. There were significant effects of the training and test phases on freezing behaviour (F (3.130, 40.691) = 11.990, P = 0.000, ε = 0.447, repeated measures ANOVA). These were characterised by freezing behaviour in the conditioning context only after footshock presentation (C1 and C2), and by conditioned freezing behaviour during the first 2 min of the two extinction trials (E1 and E2). Rats froze significantly less in the context paired with PBS infusions during extinction than in the context paired with ANI during the recall test (LTM). Thus, ANI attenuates the apparent loss of freezing behaviour produced by a prolonged 10 min exposure to a fear-conditioned context. This indicates that extinction of CFM was dependent on protein synthesis in the hippocampus. The within-subjects design of this experiment again demonstrates that two different fear memories can be independently modulated.10.1371/journal.pone.0003248.g002Figure 2Effect of ANI infusion into the hippocampus on conditioned freezing.Rats (n = 12) were fear conditioned (C1 and C2) in two distinct contexts. ANI or PBS were infused into the hippocampus immediately after extinction training by a 10 min exposure to each of the conditioned contexts at E1 and E2 such that each rat received ANI associated with one context and PBS with the other context in a counterbalanced fashion. ANI prevented the extinction of a selectively recalled fear memory because conditioned freezing measured at LTM in the context associated with ANI (E(ANI)) was greater than conditioned fear measured in the PBS associated context (E (PBS)). Results are presented as the Mean±S.E.M. *p<0.05, ***p<0.001.Extinction is correlated with increased proBDNF and decreased Arc/Arg3.1 in CA1To assess whether the extinction of contextual fear memory required BDNF in the hippocampus, ASO targeting BDNF mRNA was infused into the hippocampus 90 min prior to extinction training in one of the two conditioning contexts. Control MSO was infused before exposure to the other conditioned context (Fig. 3). During the subsequent LTM recall test, conditioned freezing behaviour was lower in the context paired with ASO infusions than in the context paired with MSO infusions. In addition, less freezing was seen in the ASO context, but not MSO context, than during extinction training. The infusion of ASO had no effect on the freezing behaviour during the extinction training sessions at E1 and E2 (Extinction×ASO×Freezing, F (1, 41) = 0.313, P = 0.579, ε = 1.000 repeated measures ANOVA), demonstrating the ASO infusions do not alter the acquisition of extinction nor change hippocampal processing non-specifically. One interpretation of these data is that MSO specifically prevents the extinction of contextual fear memory. However, this is unlikely as a NCBI BLAST search revealed that the MSO sequence does not show any homology with existing nucleotide sequences and would not act to prevent translation of any known transcript. We suggest that ASO targeting BDNF in the hippocampus promotes the extinction of contextual fear memory.10.1371/journal.pone.0003248.g003Figure 3Effect of BDNF ASO infusion into the hippocampus on conditioned freezing.BDNF ASO and BDNF MSO were infused into the hippocampus after extinction training by a 10 min exposure to two fear conditioned contexts (A and B) at E1 and E2 in a counterbalanced fashion (n = 23). BDNF ASO enhanced the extinction of a selectively recalled contextual fear memory since less conditioned freezing was seen during LTM tests in the context associated with BDNF ASO infusion (E(AS)) than the context associated with BDNF MSO infusion (E(MSO)). Results are presented as the Mean±S.E.M.. Data for the first 2 min of extinction training during E1 and E2 is shown. (F (5.401, 113.461) = 31.319, P = 0.000, ε = 0.772, repeated measures ANOVA). * P<0.05, ** P<0.01, *** P<0.001.To further elucidate the role of BDNF in the extinction of contextual fear, the levels of the BDNF-precursor, proBDNF and Arc/Arg3.1 were measured in extracts of CA1 after extinction training (Fig. 4a). Arc/Arg3.1 is a BDNF-regulated gene [29], [30], [31] that is necessary for both LTM and LTP [32], [33]. We previously showed that intrahippocampal infusions of ASO targeting BDNF prevented the increase in Arc/Arg3.1 protein associated with contextual fear conditioning and CFM [24]. We also showed that the inhibitory effects of the ASO on function were rescued by mBDNF. Recent evidence also shows that mBDNF-induced LTP in the hippocampus is mediated by Arc/Arg3.1 synthesis [34]. Together these data demonstrate a requirement for mBDNF regulated Arc/Arg3.1 in the hippocampus for the consolidation of CFM and enduring forms of plasticity. As such, measuring Arc/Arg3.1 levels in the hippocampus represents bioassay of mBDNF activity associated with CFM processing.10.1371/journal.pone.0003248.g004Figure 4Extinction training-induced changes in proBDNF and Arc/Arg3.1 protein in the CA1 of hippocampus.(a) Rats showed robust conditioned freezing during the first two min re-exposure to the training context (E) 3d after a single fear conditioning trial (C). n = 20 at C, and n = 16 at E. Following recall there was a change in proBDNF in the CA1 (F (4,14) = 8.961, P = 0.000, ANOVA). ProBDNF levels more than doubled in CA1 6 hrs after a 10 min exposure to the conditioned context (E). No changes were seen after a 2 min exposure to the fear-conditioned context (R). Arc/Arg3.1 protein in CA1 decreased 6 hrs after a 10 min exposure (E) but not following a 2 min exposure (R) to the conditioned context. (b) High levels of conditioned freezing were seen in rats administered intrahippocampal infusions of ASO and MSO 90 min before extinction training. There was no difference in the levels of freezing between the ASO and MSO administered rats at E (F (1, 7) = 4.202, P = 0.080, ANOVA). However, proBDNF levels in CA1 were altered after extinction (F (2, 9) = 6.974, P = 0.015, ANOVA) and were greater in the ASO administered when compared to control and MSO administered rats 6 hours after extinction. In the same rats, protein levels of Arc/Arg3.1 were also regulated in CA1 (F (2, 9) = 23.742, P>0.000, ANOVA), but were decreased in both MSO and ASO groups. Rats in the control group were fear conditioned at C, but were killed 3 d later. n = 4 for all groups in Western blot measurements. Results are the Mean±S.E.M. *P<0.05, **P<0.01, ***P<0.001 compared to control unless otherwise marked.Here, 48 hours after rats were conditioned to one context they underwent recall under conditions that induce either reconsolidation (2 min exposure) or extinction (10 min exposure) of CFM. A 250% increase in proBDNF in CA1 was measured 6 hours after recall conditions that produce extinction. The increase in proBDNF levels was accompanied by a 50% decrease in Arc/Arg3.1. There were no changes in proBDNF and Arc/Arg3.1, 4 or 6 hours after recall in the dentate gyrus (dg, data not shown). This agrees with cellular and molecular studies at the subregional level that show a selective role for the CA1 activity after the acquisition and retrieval of CFM [35], [36], [37]. Intrahippocampal infusions of ASO prior to a 10 min extinction trial significantly increased the levels of proBDNF protein in the CA1 6 hrs after extinction but had no effect on the decrease in Arc/Arg3.1 (Fig. 4b).These results show a direct correlation between the levels of proBDNF in the hippocampus after extinction and the magnitude of extinction of contextual fear memory. Moreover, the results also show an inverse correlation between levels of the uncleaved precursor of BDNF, proBDNF and the activity of mature BDNF in the CA1 following the extinction of CFM suggesting that extinction of long-term memories is mediated by the processing of BDNF in CA1.Extinction is correlated with Decreased Processing of BDNF in CA1To test the hypothesis that the extinction of fear memories is mediated by the proteolytic processing of proBDNF, the synthetic competitive inhibitor of tPA, tPA-STOP (2,7-bis-4(amidino-benzylidene)-cycloheptanone-1-dihydochloride) [38] was infused into the hippocampus prior to extinction training. We predicted that preventing the cleavage of proBDNF to mBDNF with tPA-STOP during extinction training would promote the extinction of contextual fear memory. Again we conditioned individual rats so that they formed two independent CFM's. The extinction of one CFM occurred after intrahippocampal infusions of tPA-STOP (Fig. 5). There was no effect of tPA-STOP on the conditioned freezing behaviour during the two extinction training phases (comparing the behaviour between the first and last two minutes of E1 with the same epochs in E2) of the experiment (Freezing×Epoch×tPA-STOP, F (1, 18) = 2.165, P = 0.158, ANOVA; Freezing×tPA-STOP interactions, F (1, 18) = 0.004, P>0.950, ANOVA). Thus suggesting that tPA-STOP has no effect on the performance during extinction training and the acquisition of extinction. However during the LTM recall tests, conditioned freezing was significantly less in the tPA-STOP-associated extinction context than in the vehicle-associated context. These results show tPA-STOP potentiated the extinction of CFM. This effect of tPA-STOP cannot be attributed to a general amnesic of the tPA inhibitor because all rats were administered tPA-STOP, but its effects on CFM were limited to the memory recalled during extinction. Furthermore, there were no affects on long-term hippocampal function because there was no evidence of a spontaneous recovery of the memory when measured one week later and ability to support a new CFM was not compromised when rats were subsequently reconditioned (Supplementary Information, \n\nFig. S2\n).10.1371/journal.pone.0003248.g005Figure 5Infusions of tPA-STOP into the hippocampus potentiate extinction of contextual fear memory.Rats (n = 11) received two 10 min extinction-training trials (E1 and E2 24 hr apart) 3 days after contextual fear conditioning in two distinct contexts (A and B). Prior to E1 they either received tPA-STOP (n = 6) or PBS (n = 5). The same rats received these compounds prior to E2 such that each rat was infused with tPA-STOP in one of the two conditioned contexts and vehicle in the other during extinction. The rats showed more conditioned freezing in the context associated with the vehicle PBS infusions than in the extinction context associated with tPA-STOP infusions during subsequent long-term memory recall tests (LTM). Results are presented as the Mean±S.E.M. Data for the first 2 min of extinction training during E1 and E2 is shown. (F (3.688, 36.88) = 35.063, P = 0.000, ε = 0.0.526, repeated measures ANOVA). **P<0.01, ***P<0.001.In addition to being an upstream activator of proBDNF cleavage, tPA has other molecular targets that may underlie the effect of tPA-STOP on extinction we report. For example, the tPA-mediated degradation of the NR1 subunit of the NMDA receptor and the extracellular matrix, as well as tPAs interaction with the low-density lipoprotein receptor related protein have been reported to influence plasticity processes in the brain [39], [40], [41], [42]. To assess whether tPA-STOP regulates proBDNF processing in extinction, proBDNF and Arc/Arg3.1 levels in CA1 were measured after extinction training (10 min recall test) following the intrahippocampal administration of tPA-STOP. Although there was a significant effect of conditioning and extinction (TEST PHASE) on freezing behaviour (F (2.079,20.788) = 45.965, P = 0.000, ε = 0.693, repeated measures ANOVA), there was no tPA-STOP X TEST PHASE interaction (F (2.079,20.788) = 0.509, P = 0.679, ε = 0.693, repeated measures ANOVA, Fig. 6a). tPA-STOP had no effect on the decrement in the fear response measured between the first and last two minutes of extinction training (“within-session” extinction of freezing). This again illustrates that tPA-STOP has no effect on the performance during extinction training, or on the acquisition of extinction. There was a significant effect of tPA-STOP on CA1 proBDNF after extinction (Fig. 6b). This was characterised by an increase in levels compared to the No Ext control rats that was further increased by tPA-STOP. Hence tPA activity regulates proBDNF levels in CA1 during the extinction of CFM. Arc/Arg3.1 was unaffected by tPA-STOP after extinction (F (2,15) = 0.562, P = 0.581, ANOVA; Levels (% No Ext); No Ext = 100±22.7, Ext-PBS = 71.3±18.4, Ext-tPA-STOP = 81.4±16.5). The increased ratio of proBDNF: Arc/Arg3.1 in CA1 under conditions of extinction further indicates decreased proBDNF processing by tPA after extinction.10.1371/journal.pone.0003248.g006Figure 6Infusions of tPA-STOP into the hippocampus potentiate the proBDNF levels in CA1 after extinction.(a) Rats (n = 18) received a single conditioning trial. 90 min prior to extinction, 3 days later, they either received tPA-STOP (n = 6) or PBS (n = 6). tPA-STOP had no effect on the decrement in the fear response measured between the first and last two minutes of extinction training. (b) There was a significant effect of tPA-STOP on CA1 proBDNF after extinction (F (2,15) = 8.003, P = 0.004, ANOVA, Fig. 6b) in the same conditioned rats 6 hr after extinction. Results are presented as the Mean±S.E.M. *P<0.01, **P<0.001 compared to No Ext group.Reconsolidation of CFM was not associated with the regulation of hippocampal BDNF or Arc/Arg3.1 levels (Fig. 4), nor requires BDNF [24]. Therefore, the long-term loss of freezing responses associated with tPA-STOP administration at recall is likely to directly reflect the impact on BDNF-mediated cellular signalling mechanisms underlying extinction rather than reconsolidation. This concurs with studies that suggest BDNF signalling is necessary for the extinction of fear memory [43], [44], but crucially indicates a role for the processing of proBDNF. Common to a number of studies that show that the extent of memory reactivation greatly influences extinction induction [45], [46], we also show that increasing the duration of context reexposure from 2 min to 10 min results in persistent, reduced conditioned freezing behaviour. It is possible that the extent of proBDNF cleavage is precisely controlled by the conditions of memory recall and that higher levels of proBDNF favour extinction as the dominant trace controlling behaviour after recall by engaging specific downstream cellular events.tPA-STOP attenuates consolidationmBDNF activity in the hippocampus is a prerequisite for the consolidation of CFM because ASO-mediated amnesia could be completely rescued by the concurrent administration of the proteolytically cleaved mBDNF protein [24]. The increased expression of Arc/Arg3.1 also suggested the activity of mBDNF was upregulated in CA1 following acquisition. Here we show that levels of proBDNF were also regulated during the consolidation of contextual fear memory (Fig. 7). Planned post hoc analyses revealed a 60% decrease in CA1 proBDNF in MSO-infused hippocampus 6 hours after contextual fear conditioning that were further reduced in ASO-infused hippocampus (Fig. 7b). Intrahippocampal infusion of ASO targeting BDNF mRNA before conditioning reduced the levels of Arc/Arg3.1 protein in the CA1 6 hours later compared to Arc/Arg3.1 measured in vehicle (PBS) and MSO infused control groups (Fig. 7c). There was no difference between Arc/Arg3.1 in CA1 in PBS and MSO groups further emphasising that the MSO used in our studies is biologically inactive. Thus we show that the levels of proBDNF decreased and the activity of mature BDNF increased in CA1 after fear conditioning. In addition, we also show amnesia-promoting ASO administration down-regulated both proBDNF and Arc/Arg3.1. These data suggest a correlation between the increased processing of proBDNF in CA1 in the formation or stabilisation of CFM.10.1371/journal.pone.0003248.g007Figure 7Fear conditioning-induced changes in proBDNF and Arc/Arg3.1 protein in the CA1 of hippocampus.(a) Rats showed conditioned freezing at LTM test 24 hrs after a single conditioning trial. n = 15 at C, and n = 3 at LTM. (b) ProBDNF decreased by half in the CA1 6 hrs after conditioning in the PBS-infused hippocampus. This was further reduced by BDNF ASO (ASO) infusions into the hippocampus prior to conditioning (F (2, 9) = 12.894, P = 0.002, ANOVA). (c) Arc/Arg3.1 protein in CA1 was selectively decreased in rats receiving BDNF ASO, but not PBS or BDNF MSO (MSO) infusions prior to conditioning. n = 4 (d) A separate group of rats (n = 11) were fear conditioned to two contexts (A and B), they received intrahippocampal infusions of tPA-STOP 90 m in before conditioning in one of the contexts and vehicle prior to training in the other. Half the rats received tPA-STOP or PBS prior to the recall test LTM 1 to determine the effect of tPA-STOP of conditioned freezing. The rats showed less post US freezing behaviour during conditioning with pretraining tPA-STOP infusions and less conditioned freezing in the drug associated context during LTM 1 compared to the control PBS context. Results are the Mean±S.E.M. (F (3.022, 30.221) = 28.352, P = 0.000, ε = 0.432, repeated measures ANOVA). ** P<0.01 compared to Pre CS, * P<0.01 compared to PBS and MSO groups for unmarked comparisons.We next investigated whether the proteolytic processing of proBDNF was causal in the formation of long-term fear memories. Rats were fear conditioned in two distinct contexts. They received intrahippocampal infusions of tPA-STOP before conditioning in one context and PBS vehicle control prior to conditioning in the other context (Fig. 7d). Half the rats also received tPA-STOP prior to the recall test at LTM1. During conditioning rats showed less post US freezing behaviour after tPA-STOP than vehicle infusion. Pre-recall test tPA-STOP had no effect on freezing behaviour during LTM1 demonstrating that tPA-STOP did not affect performance per se (F = 0.122 (1,9) P = 0.735, ANOVA). The rats also showed less conditioned freezing in the tPA-STOP-associated context than the vehicle-associated context during recall at LTM1, which did not recover 7 days later. Together these data demonstate that the acquisition of CFM is associated with increased proBDNF processing in the hippocampus.Although the consolidation of CFM is critically dependent on the mBDNF in the hippocampus, a role for proBDNF in consolidation was not previously ruled out [24]. This study shows that acquisition of CFM was correlated with a decrease in proBDNF levels in CA1. One interpretation is that decreased proBDNF-mediated signalling is also a necessary requirement for the formation of LTM. If proBDNF mediated cellular processes normally opposed consolidation, then reductions in proBDNF in the absence of changes in baseline mBDNF activity would be permissive for consolidation. However, here we show the opposite effect; infusions of ASO that prevent consolidation [24]. further reduced proBDNF levels after conditioning, while Arc/Arg3.1 levels were normalised. Thus, results from our studies are entirely consistent with a selective role for mBDNF-mediated processes in acquisition of long-term memory.DiscussionThis study provides novel insights into the molecular processes during the acquisition of long-term fear memories and those processes triggered by their selective recall. We show that reduced proteolysis of proBDNF in the hippocampus is a key regulator in protein synthesis-dependent extinction of CFM. Critically, increasing endogenous proBDNF and reducing mBDNF levels in the CA1 either with BDNF ASO or tPA-STOP, promoted extinction. Conversely, the acquisition of CFM was correlated with increased proteolytic processing of proBDNF. The demonstration of a role for BDNF in the acquisition of LTM has not been previously dissected in more chronic transgenic or pharmacological animal models. We have previously shown that consolidation but not reconsolidation of CFM is dependent on hippocampal BDNF [24]. Here we also show that conditions of recall that initiate the reconsolidation are not correlated with a change in proBDNF levels and mBDNF activity in the CA1. Therefore, the processing of BDNF was associated with the acquisition of new information and the updating of information about a salient stimulus that mediate changes in behaviour. These data generate a complete hypothesis for BDNF-associated signalling in the currently described component processes of LTM. Thus, BDNF regulates the acquisition, consolidation and extinction of fear memory, but not reconsolidation. In addition, the tPA-mediated proteolysis of proBDNF promotes new learning but opposes the extinction of established memory.The competition between extinction and reconsolidation are governed by the precise conditions of memory reactivation [45], [46]. Here we show that proBDNF cleavage is selectively inhibited under conditions of recall that favour extinction (a prolonged 10 min exposure to the context CS), but not those that promote reconsolidation (a 2 min CS presentation). This clearly demonstrates the fine control of cellular responses by ongoing experience. The differential control of the proteolysis of proBDNF by salient environmental stimuli in new learning and by learning anew after recall, also indicates the integration of new and past experience at the molecular level. Determining the molecular or cellular mechanism necessary for integrating experience will be an important endeavour. That an inhibitor of BDNF processing, tPA-STOP, can attenuate new learning but potentiate extinction, further emphasises a central role for the integration of new and past experience at the molecular level in determining future behavioral responses.This study indicates that secretion and processing of proBDNF in the adult hippocampus occurs as a consequence of memory formation. Firstly, we detected a BDNF-immunopositive signal at 35kDa (the molecular size of proBDNF) in CA1 that is specifically altered by regional infusions of ASO BDNF. This suggests that the signal is derived from the Bdnf gene. Indeed studies of CNS neurons transfected with Bdnf cDNA suggest that proneurotrophins account for a significant amount of the total neutotrophins secreted extracellularly [47], [48]. Secondly, the levels of proBDNF were regulated during consolidation and extinction. Thirdly, we showed that regional administration of tPA-STOP, an upstream inhibitor of the extracellular proteolysis of precursor BDNF [38], attenuated the processing of proBDNF in CA1. Significantly, we showed that altering the ratio of precursor to mature BDNF levels with tPA-STOP and ASO BDNF has important functional consequences for LTM. Our data concurs with other studies that have shown that several forms of long-term plasticity in the adult hippocampus were correlated with changes in BDNF processing by the extracellular protease, tPA [28], [49], [50]. It should be noted that a recent study of endogenous BDNF processing in primary cell culture has shown little, if any, proBDNF is stored and secreted from hippocampal neurons [51]. However, the failure to detect proBDNF secreted from neurons derived from embryonic tissue, in which BDNF expression is comparatively low, may consequently reflect different dynamic levels of neurortrophin transport, release and processing mechanisms to those occurring in adult neurons [26].The mechanism by which BDNF ASO potentiated the increase in proBDNF levels after extinction is unknown. It is possible that these effects may be caused by non-selective off-target, non-sequence specific effects of infusing oligonucleotides into the brain, such as the direct interaction with cellular protein or by activating immune responses [52]. However, this explanation is unlikely because the manipulation of hippocampal proBDNF protein levels and extinction of fear memory were selective for ASO and not MSO. Furthermore, the ASO and MSO had no effect on the levels of β-Actin, the not regulated reference protein used in the above experiments (data not shown). We have also previously reported effects of the BDNF ASO, but not Zif268 ASO or MSO sequences on mBDNF activity in the CA1 and the consolidation of CFM [24]. Therefore the behavioural and cellular responses to ASO are selective and are related to the targeted mRNA sequence.Protein noncoding antisense transcripts expressed from human BDNF gene locus have been identified and may function to regulate BDNF gene expression in vivo\n[53]. Therefore, it is possible that exogenous ASO infusions may interfere with the mechanism of action of endogenous antisense-BDNF to alter BDNF levels in the hippocampus. However, this explanation for the BDNF-ASO potentiated increase in proBDNF we observed is doubtful because in contrast with the human BDNF gene locus, rodent Bdnf gene loci do not encode antisense-BDNF mRNA transcripts [53], [54].Evidence from several elegant studies have suggested that opposing cellular actions of mBDNF and proBDNF mediate synaptic plasticity [55]. Namely, the cleavage of proBDNF to mBDNF by tPA is essential for LTP in the hippocampus [28]. Whilst proBDNF-mediated signalling facilitates LTD in the hippocampus via the activation of the p75 neurotrophin receptor [27]. Our evidence that hippocampal-dependent extinction is mediated by an increased proBDNF/mBDNF ratio further suggests that that the synaptic and molecular events underlying extinction closely resembles LTD [18], [20], [21], [22], [23], [56]. Our studies also show dissociable roles for mBDNF and proBDNF in the consolidation and extinction of hippocampal-dependent fear conditioning. The close correlation between the control of synaptic memory and the expression of CFM and extinction by different translational variants of BDNF, may indicate that different forms of synaptic plasticity models distinct memory processes. The precise cellular mechanism that controls the processing of BDNF by tPA required for the acquisition and extinction of long-term memory remains to be determined.The illustration that the proteolysis of proBDNF is a key regulator of two-hippocampal dependent memory processes clearly demonstrates the significant role that post-translational protein modifications (PTM) play in LTM. Recently, a mechanistic model has proposed that PTM of synaptic proteins, maintained by endogenous brain activity, play an instructive role for LTM [2]. A consequent prediction in this model is that manipulations that alter the PTM of proteins crucial for maintaining LTM cause the loss of the memory. This has recently been shown for PKMζ [57]. The model has some face validity for our data here because increased proteolysis of proBDNF was associated with the formation of LTM, while decreased processing was associated with the apparent loss (extinction) of LTM. However, we show that experimental interventions that alter the processing of BDNF are selective for the recently acquired or recalled memories, the so-called active memory [58]. Non-recalled, inactive memories were unaffected. This implies that there is a time-limited role for PTM of BDNF in LTM. In addition, since ASO targeting BDNF has no effect memory or BDNF processing after some conditions of recall (reconsolidation) [24], this suggests that the on-going maintenance of CFM is not dependent on BDNF, or the post-translational state of BDNF. This implies that BDNF is permissive for LTM by initiating the PTM of other synaptic proteins that have an instructive role in LTM, via the activation of specific signalling pathways. Future experiments are required to address this possibility.The requirement of BDNF dependent-processing for the extinction but not reconsolidation of LTM after recall suggests that drug or other interventions that directly target the PTM of BDNF, or the downstream signalling pathways of BDNF variants, potentially offers the therapeutic control of pathological memory in humans. For example, the memories that are considered to underlie phobia, post-traumatic stress disorder and drug addiction. Targeting BDNF may be particularly useful because only recalled, active, memories appear to be sensitive to manipulations that regulate with the cleavage of proBDNF to mBDNF. This has the advantage of leaving non-recalled memories intact. Furthermore, inhibiting the processing of proBDNF at recall would additionally prevent the acquisition of new memories that may be associated with the therapeutic environment and which may trigger the re-emergence of the memory by the process of renewal once away from the extinction environment [6], or cause the sensitization (augmentation) of the pathological memory [59], [60].Supporting InformationFigure S1Two separate fear memories can be independently modulated by extinction training. Five days after extinction training lower levels of conditioned freezing were measured during a recall test in the context the rats has been exposed to for 10 min during extinction training, E, than in the context that had been associated with a 2 min exposure, R, irrespective of whether context A (E(A)) or context B (E(B)) was the 10 min extinction context. Two-way repeated measures ANOVA of the freezing behaviour during T1 revealed an Extinction Training X Context interaction (F = 12.476 (1,10), p = 0.005), but no significant effect of Context (F = 0.024 (1,10), p = 0.897). Results are presented as the Means.(6.01 MB TIF)Click here for additional data file.Figure S2Infusions of tPA-STOP into the hippocampus potentiate extinction of contextual fear memory and have no long-term effect on hippocampal function. As described previously (Fig. 5), rats (n = 11) received two 10 min extinction-training trials (E1 and E2 24 hr apart) 3 days after contextual fear conditioning in two distinct contexts (A and B). Prior to E1 they either received tPA-STOP (n = 6) or PBS (n = 5). The same rats received these compounds prior to E2 such that each rat was infused with tPA-STOP in one of the two conditioned contexts and vehicle in the other during extinction. Rats showed more conditioned freezing in the context associated with the vehicle PBS infusions than in the extinction context associated with tPA-STOP infusions during long-term memory recall test (LTM 1) 1 day after extinction. Additionally, this data showed that there was no spontaneous recovery of the fear memory measured at a subsequent recall test 7 days later. All rats were re-conditioned in one context (A or B). A recall test was performed in both the contexts 1-2 days later (LTM3). At LTM3 rats showed significantly higher levels of conditioned freezing in the reconditioned context (C3) than in the context not associated with reconditioning (no C3). This indicates that (i) tPA-STOP has no long-term affect on hippocampal function because the rats can support anew a contextual fear memory for a specific context. 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+{
+    "id": "PMC2532759",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2532759\nAUTHORS: Jeanne Farrell, Lavoisier Ramos, Martin Tresguerres, Margarita Kamenetsky, Lonny R. Levin, Jochen Buck\n\nABSTRACT:\nBackgroundMammalian Soluble adenylyl cyclase (sAC, Adcy10, or Sacy) represents a source of the second messenger cAMP distinct from the widely studied, G protein-regulated transmembrane adenylyl cyclases. Genetic deletion of the second through fourth coding exons in Sacytm1Lex/Sacytm1Lex knockout mice results in a male sterile phenotype. The absence of any major somatic phenotype is inconsistent with the variety of somatic functions identified for sAC using pharmacological inhibitors and RNA interference.Principal FindingsWe now use immunological and molecular biological methods to demonstrate that somatic tissues express a previously unknown isoform of sAC, which utilizes a unique start site, and which ‘escapes’ the design of the Sacytm1Lex knockout allele.Conclusions/SignificanceThese studies reveal increased complexity at the sAC locus, and they suggest that the known isoforms of sAC play a unique function in male germ cells.\n\nBODY:\nIntroductionIn mammals, the widely studied second messenger cAMP can be generated by two types of enzymes: G protein-regulated transmembrane adenylyl cyclases (tmACs) and bicarbonate-regulated soluble adenylyl cyclase (sAC). Nine distinct genes encode a family of tmAC isoforms which display differential tissue distribution and responsiveness to calcium. Each tmAC isoform is modulated by heterotrimeric G proteins in response to hormones and neurotransmitters (reviewed in [1]). In contrast, a single sAC gene [2] generates multiple isoforms by alternative splicing [3], [4] whose activities are directly stimulated by bicarbonate and calcium ions [5]–[8]. A second sAC-related locus present in human, dog and other mammalian genomes, but not detected in mouse or rat genomes, appears to be a pseudogene [9].The sAC protein was initially purified from rat testis cytosol, and two independent cDNAs, which were subsequently shown to represent alternatively spliced isoforms [4], were cloned from a rat testis cDNA library [2]. These two transcripts were termed full-length (sACfl), encoding a 187 kD protein, and truncated (sACt), encoding a 53 kD protein (Fig. 1A). The protein originally purified corresponds to sACt. This isoform is highly active but of relatively low abundance. We required approximately 1000 rat testis to recover sufficient material to obtain sequence information [2], [10], and detecting sACt in testis cytosol from wild type mice by Western blotting required an initial enrichment step; i.e., immunoprecipitation with a different sAC-specific monoclonal antibody [11]. The majority of immune reagents generated, protein biochemistry and kinetics, and the design of a knockout mouse have been based on the knowledge of the sACt and sACfl isoforms.10.1371/journal.pone.0003251.g001Figure 1Schematic organization of (A) previously identified, testicular sAC transcripts and (B) the newly identified somatic sAC transcript.Boxes denote exons. C1 and C2 refer to the two catalytic domains. Red exons contain stop codons. (A) sACfl is encoded by all known coding exons (32), and sACt is generated by skipping exon 12. Yellow exons (2-4) are removed in the Sacytm1Lex allele. Arrows indicate approximate locations of epitopes for the indicated monoclonal antibodies (R40, R21, and R37). (B) Somatic sAC transcripts derive from a unique start site upstream of exon 5 and continue through at least exon 16 to an unknown stop.Historically, ‘soluble’ adenylyl cyclase activity had only been detected in testis cytosol [12], [13]. Initial Northern blot data confirmed that sAC message is abundant in testis [2], and that it is specifically enriched within the developing male germ cells [14]. But more sensitive methods of mRNA detection, including RT-PCR [14] and multiple tissue expression arrays [15], revealed sAC mRNA to be universally expressed. For example, the NCBI Gene Expression Omnibus database chronicles sAC expression in a number of somatic tissues, including brain. Finally, the GNF gene expression Atlas and in situ analysis performed by the Allen Brain Institute identified sAC message throughout the nervous system including dorsal root ganglia, spinal cord, cerebellum, hypothalamus, and thalamus [16].To examine sAC protein expression, we and others, have raised various polyclonal antisera and numerous monoclonal antibodies against sAC [3], [4], [6], [17], [18]. These immune reagents predict sAC to also be expressed in a large number of cell lines [3], [18] and a variety of somatic tissues [6], [17], [19]–[24] However, the sAC protein identified in cells and tissues tends to be associated with intracellular organelles [18], [24], [25] or vesicles [20], implying that somatic sAC is not a soluble protein but could require detergent extraction.Somatic functions for sAC are predicted by both genetic and pharmacologic experiments. The human sAC locus was implicated in familial absorptive hypercalciuria (AH) [15], a syndrome of calcium homeostasis defects in intestine, kidney and bone. Pharmacological methods taking advantage of sAC-selective versus tmAC-selective inhibitors have identified a role for sAC as a cellular sensor of pHi in epididymis [19] and kidney [20], a CO2/HCO3 sensor in airway cilia [21], a mediator of oxidative burst in response to tumor necrosis factor in human neutrophils [26], and a modulator of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in corneal endothelium [22] and in human airway epithelium [17]. In certain isolated primary cells and cell lines, we have been able to use sAC-specific RNAi mediated knockdown to confirm results obtained with sAC- and tmAC-selective pharmacological inhibitors. Using these more stringent criteria, we have elucidated additional roles for sAC in neuronal responses to the guidance cue Netrin-1 [23] and in cellular responses to the neurotrophin, Nerve Growth Factor (NGF) [27], [28].These numerous putative somatic functions for sAC are inconsistent with the initial descriptions of a very specific germ cell phenotype in the existing sAC knockout (Sacytm1Lex/Sacytm1Lex) mouse [11], 29, 30. These mice were generated by homologous recombination with an exon trapping, IRES-lacZ expression cassette replacing the 2nd through 4th coding sequence exons present in both sACt and sACfl isoforms [29]. In mice with the Sacytm1Lex locus, lacZ expression was only detected in testis, suggesting that at least the promoter and exon 1 are specific to male germ cells. More importantly, homozygous male knockout mice are sterile; their sperm are immotile, do not undergo capacitation, and are unable to fertilize an egg in vitro\n[11], [29]. A more extensive phenotypic analysis revealed that female Sacytm1Lex/Sacytm1Lex mice display increased circulating cholesterol and triglyceride levels and both male and female homozygous knockout animals have slightly elevated heart rates [as deposited in the Mouse Genome Database [31]]. And even though knockout phenotypes are often muted or absent due to compensation, such subtle somatic phenotypes are unexpected considering the variety of physiological functions demonstrated or predicted for sAC. For example, even though we demonstrated that sAC is essential for Netrin-1 induced axonal outgrowth in commissural axons [23], Sacytm1Lex/Sacytm1Lex mice do not exhibit the pronounced structural brain defects [23], [30] seen in Netrin-1 knockout animals [32].Moe and co-workers cloned human sAC cDNAs from somatic cells whose open reading frames do not contain exons deleted in Sacytm1Lex/Sacytm1Lex mice [3]. If such cDNAs represent the predominant species of sAC in mammalian somatic tissues, it would explain how the Sacytm1Lex knockout could exhibit exclusively a germ cell phenotype. Here we use immunological and molecular methods to confirm the existence of previously unknown somatic isoforms of sAC. These somatic sAC isoforms derive from a unique mRNA start site which “escapes” the design of the Sacytm1Lex mouse.ResultssAC-specific antibodies identify isoforms unaffected in Sacytm1Lex ‘knockout’We used two sAC-specific monoclonal antibodies recognizing distinct, non-overlapping epitopes to examine the molecular nature of sAC proteins expressed in brain. R21 is a monoclonal antibody recognizing an epitope in coding sequence exon 5 (within amino acids 206–216) of sACfl while R37 is a monoclonal antibody recognizing an epitope in exon 11 (within amino acids 436–466) (Fig. 1). Due to compelling evidence for a function of sAC in neuronal signaling [23], [27] and because we previously demonstrated we could recover sAC activity (i.e., it was inhibited by the sAC-specific inhibitor, KH7, and it was insensitive to the tmAC activator, forskolin) by R37 immunoaffinity purification from detergent extracts of rat brain [23], we first focused on the sAC proteins present in mouse brain. Western blots using R21 revealed a number of immunoreactive bands. Surprisingly, none of these putative sAC bands were altered in the Sacytm1Lex/Sacytm1Lex mice (Fig. 2A, first two lanes).10.1371/journal.pone.0003251.g002Figure 2Somatic sAC isoforms unaffected in Sacytm1Lex locus.Immunoprecipitations (IP) using mAb R37 or IgG control antibody from detergent solubilized whole cell extracts (lysates) of brains (A,B) or kidney (C) from wild type or Sacytm1Lex/Sacytm1Lex mice were subjected to Western analysis using (A,C) biotinylated R21 mAb (R21B) or (B) biotinylated R37 mAb (R37B). White circles denote nonspecific bands detected with streptavidin (no primary antibody) alone. The smear at ∼50 kDa in the R37 IP from brain resolves to at least two bands when less of the IP is loaded for Western blotting (inset).We also examined the sAC activity immunoprecipitated by R37 from wild type and Sacytm1Lex/Sacytm1Lex brains (Fig 3). We previously showed the adenylyl cyclase activity immunoaffinity purified from wild type mouse brains was insensitive to forskolin and inhibited by sAC-specific inhibitor [23]. Preliminary analysis suggests that there is considerably less sAC adenylyl cyclase activity immunoprecipitated from a single wild type brain (77 pmol cAMP/ml) than from wild type testis (at least 254 pmol cAMP/ml) (Fig. 3A). This is not surprising considering sAC mRNA expression in testis is greater than in any somatic tissue, including brain [14], and because testis expresses the highly active, sACt isoform [5], [11]. Equivalent amounts of adenylyl cyclase activity were immunoprecipitated from wild type or Sacytm1Lex/Sacytm1Lex brains (Fig. 3B), and the activity from Sacytm1Lex/Sacytm1Lex brains was confirmed to be sAC by its insensitivity to forskolin (no statistically significant difference in the presence or absence of 10 μM forskolin) and its sensitivity to the sAC-selective inhibitor, 4-hydroxyestradiol. The sAC-selective catechol estrogen, 4-hydroxyestradiol [19], [33], [34], was used to inhibit the immunoprecipitated activity from Sacytm1Lex/Sacytm1Lex brains because it is unaffected by the detergents used during immunoprecipitation. The immunoprecipitated activity from Sacytm1Lex/Sacytm1Lex brains was inhibited by approximately 50% in the presence of catechol estrogen (Table 1). These data suggested that the sAC protein(s) in brain was unaffected by the deletion of exons 2-4, and would therefore represent novel, previously uncharacterized isoforms.10.1371/journal.pone.0003251.g003Figure 3Somatic sAC activity in brain is lower than activity in testis, but it is not diminished in brains from Sacytm1Lex/Sacytm1Lex mice.(A) Adenylyl cyclase activity (in pmol cAMP formed per ml) in mouse IgG or R37 IP from detergent extracts from a single mouse brain or mouse testis from wild type mice. Activity from testis may be under-represented; we did not confirm antibody was in excess. MM is adenylyl cyclase reaction conditions alone (no IP added). Control IgG activity is derived from pooled brain and testis detergent extracts. Values represent averages of duplicate determinations. (B) Adenylyl cyclase activity in R37 IPs from wild type (WT) or Sacytm1Lex/Sacytm1Lex (KO) mice. MM is adenylyl cyclase reaction conditions alone (no IP added). Extracts were precleared through mouse IgG prior to immunoprecipitation. Values represent quadruplicate determinations from two wild type and two knockout brains with error bars indicating S.E.M.10.1371/journal.pone.0003251.t001Table 1sAC activity from Sacytm1Lex/Sacytm1Lex brainscAMP* (pmol/mL)Standard Deviation (pmol/mL)Basal activity20.4724.1384-hydroxyestradiol (100 μM)11.2230.726*Average of quadruplicate determinations.To determine whether the immunoreactive bands were recognized by both antibodies, which would provide a compelling argument that they are bona fide sAC isoforms, we immunoprecipitated using R37 followed by Western blotting with R21 (biotinylated R21 was used for Western blotting to prevent detection of the immunoprecipitating R37 IgG). Western blotting the R37 immunoprecipitates reveals that at least two of the immunoreactive bands, at approximately 50 kDa, are recognized by both sAC-specific monoclonal antibodies (Fig. 2A,B). These two proteins are also diminished in the brain extracts following specific (R37) immunoprecipitation, but remain in brain extracts following immunoprecipitation with control (isotype-matched IgG) antibody. Thus, we have identified two proteins of approximately 50 kDa proteins, which are recognized by distinct sAC-specific monoclonal antibodies and are correlated with sAC-like adenylyl cyclase activity. Yet, both isoforms are unaffected in brains from Sacytm1Lex/Sacytm1Lex mice.If these brain sAC isoforms were unaffected by removal of exons 2-4, they should not be recognized by antisera directed against this region. One of our monoclonal antibodies, R40, recognizes an epitope which spans exons 2 and 3 (Fig. 1A). Using R40, we were able to immunoprecipitate a 50 kDa protein from wild type mouse testis cytosol, which was absent in testis cytosol from Sacytm1Lex/Sacytm1Lex mice (Fig. 4, top). This 50 kDa isoform is presumably sACt. Not only was this isoform not detectable in brain, but this exon 2-3 directed monoclonal antibody did not immunoprecipitate any detectable sAC isoforms from wild type or Sacytm1Lex/Sacytm1Lex brains (Fig. 4, bottom). The testis cytosolic isoform, sACt, and one of the newly identified detergent extractable, brain isoforms run together at ∼50 kDa and are not easily distinguished on SDS/PAGE (compare Fig. 2 and 4). We believe this coincidence has contributed to previous confusion about the molecular identity of sAC isoforms identified by Western blotting.10.1371/journal.pone.0003251.g004Figure 4Sperm sAC isoforms are not detected in brain.Immunoprecipitations (IP) using mAb R40 or isotype-matched IgG control antibody from detergent solubilized whole cell extracts (lysates) of testis (top) or brain (bottom) from wild type or Sacytm1Lex/Sacytm1Lex mice were subjected to Western analysis using biotinylated R21 mAb. The sharp band at ∼50 kDa in the R40 IP from wild type testis is distinct from the faint, background bands found in Sacytm1Lex/Sacytm1Lex mice and in the control IgG IP.We next asked whether the detergent extractable, 50 kDa isoforms present in brain could be found in other somatic tissues. In kidney, sAC has been proposed to form a complex with the vacuolar proton ATPase to regulate renal distal proton secretion [20]. Western blots (using R21) of R37 immunoprecipitates from detergent extracts of wild type and Sacytm1Lex/Sacytm1Lex kidneys revealed a ∼50 kDa sAC isoform, which once again, was unaffected by removal of exons 2-4 (Fig. 2C). These data suggest that at least one of the two ∼50 kDa brain isoforms represents a widely distributed, somatic isoform of sAC.sAC transcripts in brain use an alternate start siteWe used the knowledge that brain sAC isoforms must contain the epitopes recognized by R21 (amino acids 206–216) and R37 (amino acids 436–466) to explore the nature of brain sAC cDNAs. Using primers specifically recognizing these sequences and 35 cycles of amplification, we detected a fragment in brain mRNA from wild-type mice; with forty rounds of amplification, we amplified fragments from both wild type and Sacytm1Lex/Sacytm1Lex brains (Fig. 5). In each case, nucleotide sequencing confirmed the amplified fragment contained a single product corresponding to the complete sequence (i.e., contained all known exons) between exons 5 and 11. The need for greater than 30 rounds of amplification to detect sAC message in somatic tissues is consistent with the recently published identification of sAC mRNAs in specific neuronal cell types [30]. PCR amplification of the LacZ/Neo cassette confirmed the identity of Sacytm1Lex/Sacytm1Lex tissues, but it is unclear why knockout brains and testis appear to express equivalent LacZ/Neo message.10.1371/journal.pone.0003251.g005Figure 5RT-PCR of cDNA from testis and brain from wild type (WT) and Sacytm1Lex/Sacytm1Lex mice (KO).(A) PCR across exons 15-16. (B) PCR across exons 5-11. (C) PCR for β-actin loading control. (D) PCR for LacZ/Neo. (−) is a no template control. The number in the lower right corner of each panel is the number of cycles used in each experiment.Consistent with the facts that R40 did not detect any sAC isoforms in brain and the isoforms we do detect in brain are unaffected by removal of exons 2-4, we were unable to amplify a product using exon 1, 2, 3, or 4 sense primers to exon 5 antisense primers from either wild type or knockout brain mRNA (Fig. 6 and data not shown). As control, the exon 1 sense and exon 5 antisense primers amplified the expected size product using testis mRNA from wild type and Sacytm1Lex/+ heterozygous mice (Fig. 6). These primers also amplified a smaller product (of 200 base pairs) from Sacytm1Lex/+ heterozygous and Sacytm1Lex/Sacytm1Lex homozygous knockout mice which nucleotide sequencing confirmed to be an in-frame, exon1:exon5 spliced product arising exclusively from the Sacytm1Lex allele. It is possible this uniquely spliced product in testis from knockout mice is responsible for the residual adenylyl cyclase activity identified in sperm from Sacytm1Lex/Sacytm1Lex mice [35]. It is also possible this aberrant, testis-specific product diminishes splicing into the LacZ/Neo cassette, explaining why the level of LacZ/Neo message in testis is not much higher than the levels found in brain (Fig. 5).10.1371/journal.pone.0003251.g006Figure 6PCR from Exons 1 through 5 from WT and knockout mouse tissues.(A) and (C) PCR across exons 1-5. (A) Testis first strand from WT (+/+), Sacytm1Lex/+ heterozygote (+/−), and Sacytm1Lex/Sacytm1Lex homozygous knockout (−/−) mice. (C) First strand cDNA from brain (B), heart (H), kidney (K), or liver (Li) from Sacytm1Lex/Sacytm1Lex homozygous knockout (−/−) mice; (−) indicates no template control. (B) and (D) β-actin controls. The number in the lower right corner of each panel is the number of cycles used in each experiment.To test whether brain sAC mRNAs use an alternate start site, we performed 5′ Rapid Amplification of cDNA Ends (RACE) starting in exon 5. We obtained two 5′ RACE products, which extended beyond the intron/exon boundary of exon 5 into the sequence previously thought to be intron. The two fragments were contiguous; the larger fragment simply extended further (344 nucleotides) into the intron upstream of coding exon 5 (Fig. 7; GenBank Accession #EU478437). Neither fragment extended into sequences removed in the Sacytm1Lex locus, and these upstream sequences did not correspond to any of the alternatively spliced transcripts identified in human tissues [3]. Thus, sAC mRNAs in mouse brain appear to utilize a unique start site, which would not be deleted in Sacytm1Lex/Sacytm1Lex mice (Fig. 1B).10.1371/journal.pone.0003251.g007Figure 7Sequence of 5′ RACE product defining new mRNA start site from mouse brain.Sequence in bold is the newly defined 5′UTR which corresponds to the region previously assigned to be the intron between Exons 4 and 5.We had first concluded that sAC is widely distributed in mammals based upon an RT-PCR experiment using primers in exons 15 and 16 [14]. Reed et al. reached a similar conclusion using an overlapping region as probe in a multiple tissue array blot [15]; therefore, exon 16 is likely included in somatic isoforms of sAC. Using a sense primer corresponding to the newly identified 5′ end and an antisense primer in exon 16, we amplified a single product, from both wild type and Sacytm1Lex/Sacytm1Lex mouse brain mRNA, which extended from the newly described start site through exons 5 to 16 (Fig. 1B). This cDNA was amplified in both wild type and Sacytm1Lex/Sacytm1Lex brains. While we still do not know the true 3′ end of brain sAC cDNAs, we expect them to be significantly shorter than sACfl because the encoded protein isoforms are only ∼50 kDa. Consistent with this, we were unable to amplify a product from the new start site to exon 32 (data not shown). This new mRNA start site, outside the region deleted in the Sacytm1Lex locus, predicts a previously unappreciated promoter used for expression of sAC in mouse brain. Because sAC proteins in mouse brain derive from this alternate promoter, downstream from the deleted exons and inserted IRES-lacZ cassette, one would not expect to find LacZ expression in brain nor a neuronal defect in Sacytm1Lex/Sacytm1Lex mice.DiscussionThese findings reveal the sAC locus to be more complex than previously appreciated. The biochemically characterized sACt and sACfl isoforms may ultimately prove to be predominantly, if not exclusively, expressed in male germ cells, explaining the relatively specific male sterile phenotype of Sacytm1Lex/Sacytm1Lex mice [11], [29]. In contrast, the presumptive promoter identified here, which directs expression of brain mRNAs initiating upstream of coding exon five, is likely to direct expression of sAC in many somatic tissues. Thus, the relatively subtle somatic phenotypes reported for Sacytm1Lex/Sacytm1Lex mice [31] may be due to altered expression of a somatic sAC isoform instead of loss of sACt or sACfl. Consistent with this hypothesis, we routinely required increased rounds of PCR amplification to detect sAC messages from Sacytm1Lex/Sacytm1Lex tissues (Fig. 5).Somatic isoforms encoded by transcripts from this promoter will possess only the second (C2) of the two identified catalytic domains [2]. Heterologous expression of C2-only containing isoforms has thus far failed to result in adenylyl cyclase activity [3], so how these isoforms produce cAMP remains an open question. By possessing only the C2 catalytic domain, somatic sAC isoforms will differ from the previously characterized, C1-C2 containing isoforms which may be exclusively expressed in male germ cells. Thus, it would be possible to design safe and effective contraceptive strategies by identifying inhibitors selective for C1-C2 containing sAC isoforms.The evolutionary conservation of bicarbonate-mediated cAMP generation across many kingdoms [6], [36]–[39], including from the earliest known forms of life, Cyanobacteria [6], [36], suggests that this signal transduction pathway should be fundamentally important in biology. For example, multiple physiological processes, in addition to sperm function, are modulated by CO2 and/or HCO3\n− (i.e., diuresis, breathing, blood flow, cerebrospinal fluid and aqueous humor formation) [40]. In most cases, the effects of CO2/HCO3\n− have been ascribed to as yet undefined chemoreceptors [40]–[42], but potential links to cAMP signaling, such as in carotid body [43], suggest additional, somatic roles for a bicarbonate regulated adenylyl cyclase, such as sAC. With our identification of novel isoforms of sAC in somatic tissues, such additional roles remain possible.Materials and MethodsAnimals2–4 month old wild type or Sacytm1Lex/Sacytm1Lex mice [29] were euthanized with CO2, and brains, kidneys, or testis were immediately dissected, flash frozen in liquid N2 and stored at −80°C until processing. All animal work was performed with approval from the Institutional Animal Care and Use Committee of Weill Cornell Medical College (IACUC Protocol #0604-487A).Immunoprecipitation from detergent extractsBrains or kidneys were thawed and homogenized in detergent lysis buffer in the presence of protease inhibitors (50 mM Tris, 150 mM NaCl, 0.4 mM EDTA, 0.1 mM DTT, 1 M PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1% NP40) (1∶10 w/v). All steps were performed on ice (or at 4°C) unless specified. Homogenates were centrifuged at 45,000xg for 50 minutes. The protein concentration of the supernatant fraction was determined (BioRad) and an aliquot saved at 4°C for Western analysis (‘pre-IP lysate’). Equivalent protein amounts from different supernatants were precleared by incubation with protein G beads (Amersham Pharmacia) (100 μl beads/100 mg total protein) overnight at 4°C. Samples were centrifuged at full speed in an Eppendorf centrifuge for 10′, and the supernatant was collected into fresh tubes. Clarified lysates were incubated with specific anti-sAC antibodies (R37 or R40) or control, mouse IgG at a concentration of 20 μg antibody/mg protein for 4 h at 4°C. Immune complexes were collected on Protein G beads (50 μl/100 mg total protein) and incubated for 1 h. Beads were collected by centrifugation, and an aliquot of the supernatant was collected for Western analysis (post-IP supernatant). Beads were washed three or four times in detergent-free lysis buffer.For Western analysis, beads were incubated in SDS/PAGE sample buffer (BioRad) containing 5% β-mercaptoethanol for 5′ at room temperature, briefly spun, and an aliquot used for SDS/PAGE. Proteins were transferred to PVDF membranes, which were blocked in 5% milk (BioRad) for 1 hour at room temperature, rinsed once with TBST and incubated with biotinylated mAbs, R21 or R37 (1∶500 in TBST) overnight at 4°C. Control blots to examine streptavidin binding proteins were incubated in TBST alone. Membranes were rinsed in TBST (4×15′) and incubated with a HRP-conjugated streptavidin (1∶5000 in TBST) for 1 hour at room temperature. Bands were visualized using enhanced chemiluminescence (Pierce Co.)For activity assays, beads were incubated in 100 μl reaction buffer containing 200 mM Tris pH 7.5, 100 U/ml phosphocreatine kinase, 20 mM creatine kinase, 2.5 mM ATP, and 10 mM MgCl2 for 30 minutes at 30°C. Where indicated, reactions contained 10 μM forskolin or 100 μM 4-hydroyestradiol (Steraloids, Inc.) or equivalent volumes of vehicle. Reactions were terminated by adding reaction supernatant into 100 μl 0.2 M HCl, samples were neutralized according to manufacturers protocol and cAMP quantitated using Correlate-EIA Direct Assay (Assay Designs, Inc).\nAntibody epitope mapping\nsACt sequence was first divided into 11 fragments, and primers were designed to amplify all 11 fragments, with forward primer 5′CACC overhang for cloning into pENTR/D-TOPO entry vector (Gateway System, Invitrogen) followed by ATG, where necessary. The entry clone was subsequently recombined with pDEST15 vector to create 11 sAC sequence fragments with N terminal GST tag. The N terminal tag was necessary to monitor protein expression and to increase fragment size to facilitate analysis by SDS-PAGE. Clones were shuttled into BL21-AI cells, and expression induced by addition of final concentration of 0.2% L-arabinose (Sigma) for 3–4 hours. Pelleted bacteria was resuspended in Laemmli sample buffer, run on SDS-PAGE and immunoblotted with each monoclonal antibody of interest. To narrow down the antibody epitope, forward and reverse complimentary primers encoding 14–17 amino acid stretches were designed to cover each of the recognized fragments. Each large fragment was covered by 5 overlapping smaller fragments. Forward primers contained CACC overhangs and N terminal ATG for cloning into pENTR/D-TOPO vector. Complimentary oligomers were annealed by incubation in cooling water in the presence of buffer containing 50 mM NaCl, 10 mM Tris-HCl pH 8, 10 mM MgCl2, 1 mM DTT, cloned into the entry vector, and recombined to generate GST-fusion proteins. Proteins were expressed and epitopes were defined by Western blotting using each monoclonal antibody.RNA production, and RT-PCR amplification of sAC productsTissues harvested from wild type or Sacytm1Lex/Sacytm1Lex mice were immediately placed in Trizol and either stored at −80°C or processed for total RNA according to manufacturer's protocol. Total RNA was quantified spectrophotometrically, and at least 2 mg of total RNA was used to generate polyA+ RNA using the Micro Poly(A) Purist Kit according to manufacturer's protocol (Ambion). Purified polyA+ RNA was resuspended in DEPC-treated water and treated with amplification grade DNase I according to the manufacturer's protocol (Invitrogen). DNase-free polyA+ RNA was stored at a final concentration of approximately 100 ng/mL at −80°C until use.Approximately 500 ng of polyA+ RNA was used to generate first strand cDNA using Invitrogen's Platinum Taq PCR kit according to manufacturer's instructions. Briefly, RNA was incubated with 50 mM oligo(dT)20, (or 10 mM gene specific primer), 10mM dNTP and DEPC-treated water in a volume of 10 μl for 5 minutes at 65°C. An equal volume of cDNA Synthesis Buffer was added, yielding final concentrations of 1× Reverse Transcriptase Buffer (Invitrogen), 10 mM DTT, 125 mM MgCl2, 40U RNaseOUT, 200U SuperScript III Reverse Trancriptase, and the reaction was incubated for 60 minutes at 50°C. The reaction was terminated by incubation at 85°C for 5 minutes, and placed on ice. 1μl of RNase H (2 U/ml) was added, and incubated at 37°C for 20 minutes. This first strand was stored as single use aliquots at −80°C until use.Routinely, PCR reactions used a standard three step protocol using Platinum Taq (Invitrogen, Inc.) with an initial denaturation step at 93°C for 3 minutes, followed by 35 or 40 cycles of 93°C for 20 seconds, 60°C for 20 seconds, and 68°C for 1 minute, followed by a final step at 68°C for 10 minutes. In wild type somatic tissues, 35 cycles was sufficient to detect sAC amplified products, but Sacytm1Lex/Sacytm1Lex somatic tissues routinely required 40 rounds of amplification to detect fragments.Primers used to amplify from exons 1 to 5:Forward: LRL1127: 5′-ATGAGTGCGCGAAGGCAGGAAT-3���\nReverse: LRL1511: 5′-CTGCTCTCTGATCTGGAATCCTC-3′\nPrimers used in to amplify from new mRNA start site to exon 16:Forward: Up5: 5′-ACCCAGAATGTGTTGTGCAAAC-3′\nReverse: LRL1519: 5′-CTTGTCCCGGATTTCCTGAGGCTG-3′\nPrimers used in to amplify from exons 15 to 16:Forward: LRL1518: 5′-CAGAAGCAACTGGAAGCCCTG-3′\nReverse: LRL1519: 5′-CTTGTCCCGGATTTCCTGAGGCTG-3′\nPrimers used to amplify the βGal/Neo cassette:Forward: LRL 1276: 5′-GGAACTAACAGAGATCTATCTGC-3′\nReverse: LRL 1277: 5′-GGATGGACCATCTAGAGACTGCCA-3′\nPrimers used to amplify β-actin:Forward: BF: 5′-GGAGAAGATCTGGCACCACAC-3′\nReverse: BR: 5′-GGTGACCCGTCTCCGGAGTCC-3′\n\n5′ Rapid Amplification of cDNA Ends (5′RACE)\n5′RACE was performed on 500 ng of polyA+ RNA from brains of sACytm1Lex/sACytm1Lex mice using 5′RACE Kit (Invitrogen) according to the manufacturer's protocol.5′RACE Primers used from Exon 5:Ex5GSP1: 5′-CTGCTCTCTGATCTGGAATCCTC-3′\nEx5GSP2: CAATTTCAATCATGCTCCGATCACAG\nEx5GSP3: CAGCAGTTTGGTGACAAAATAACGTCG\n\nREFERENCES:\n1. 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batch_11/PMC2533019.json

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+{
+    "id": "PMC2533019",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2533019\nAUTHORS: Eva Bellemain, Eldredge Bermingham, Robert E Ricklefs\n\nABSTRACT:\nBackgroundThe bananaquit (Coereba flaveola) is a small nectivorous and frugivorous emberizine bird (order Passeriformes) that is an abundant resident throughout the Caribbean region. We used multi-gene analyses to investigate the evolutionary history of this species throughout its distribution in the West Indies and in South and Middle America. We sequenced six mitochondrial genes (3744 base pairs) and three nuclear genes (2049 base pairs) for forty-four bananaquits and three outgroup species. We infer the ancestral area of the present-day bananaquit populations, report on the species' phylogenetic, biogeographic and evolutionary history, and propose scenarios for its diversification and range expansion.ResultsPhylogenetic concordance between mitochondrial and nuclear genes at the base of the bananaquit phylogeny supported a West Indian origin for continental populations. Multi-gene analysis showing genetic remnants of successive colonization events in the Lesser Antilles reinforced earlier research demonstrating that bananaquits alternate periods of invasiveness and colonization with biogeographic quiescence. Although nuclear genes provided insufficient information at the tips of the tree to further evaluate relationships of closely allied but strongly supported mitochondrial DNA clades, the discrepancy between mitochondrial and nuclear data in the population of Dominican Republic suggested that the mitochondrial genome was recently acquired by introgression from Jamaica.ConclusionThis study represents one of the most complete phylogeographic analyses of its kind and reveals three patterns that are not commonly appreciated in birds: (1) island to mainland colonization, (2) multiple expansion phases, and (3) mitochondrial genome replacement. The detail revealed by this analysis will guide evolutionary analyses of populations in archipelagos such as the West Indies, which include islands varying in size, age, and geological history. Our results suggest that multi-gene phylogenies will permit improved comparative analysis of the evolutionary histories of different lineages in the same geographical setting, which provide replicated \"natural experiments\" for testing evolutionary hypotheses.\n\nBODY:\nBackgroundWe previously used mitochondrial DNA analyses to describe the history of population expansion and divergence of the bananaquit (Coereba flaveola), the most abundant and widely distributed songbird in the West Indies, which also occurs in the humid tropics of continental America [1-3]. The geographic distribution and relationship of mtDNA RFLP haplotypes suggested that populations of this species were periodically invasive, and then quiescent. Here we increase our sample of populations and genes to dissect more finely the population history of bananaquits.The bananaquit is a small nectivorous and frugivorous emberizine bird (order Passeriformes) that is an abundant resident throughout the West Indies, except for Cuba [4-6]. On the continent, it is widely distributed from southern Mexico through much of South America. Geographic variation in plumage coloration led to the recognition of 41 subspecies throughout the range of the bananaquit [7], but mtDNA RFLP analysis failed to support many of these distinctions [3]. The mtDNA analyses also revealed levels of genetic divergence between some populations within the range of genetic differences observed between species [8,9].Building on our earlier studies of the bananaquit [1-3], we have now included representatives of more populations from the West Indies and the continent, expanded our nucleotide sample of the mitochondrial genome of each individual to 3744 base pairs, and have added at least 2049 base pairs representing three nuclear gene regions. Our phylogenetic analysis is rooted by the addition of five individuals of three species of emberizid finches representing three genera.Our principal objectives were to determine the ancestral area of C. flaveola, to infer the number and directions of colonization events within the West Indies and between the islands and the continent, and to assess the power of nuclear versus mitochondrial genes to inform analyses of colonization history and subsequent population dynamics. Our phylogenetic results locate the geographic ancestral area of the bananaquit in the region of the Greater Antilles and Bahamas, and strongly support the hypothesis that continental bananaquits were derived from West Indian ancestors. In addition, comparisons of mitochondrial and nuclear gene genealogies exposed a complex and dynamic history of bananaquits in the Greater and Lesser Antilles that would have remained masked without recourse to both maternal and biparental genealogical markers.ResultsSequencing, nucleotide composition and saturationWe obtained clean sequences for all 43 C. flaveola distributed within the West Indies and South-central America (Figure 1) and for six outgroup samples, with a few exceptions, for six mitochondrial genes and three nuclear genes (see additional file 1). All sequences were submitted to Genbank (accession numbers from EF567429 to EF567954; additional file 1).Figure 1Geographic representation of the sample localities of the Coereba flaveola specimens analyzed. Locality codes are the same as in additional file 1.Cloning was performed on nine samples for BFib5, 10 samples for Rag1, and two samples for CHDZ. Cloning error rate was estimated to be about 0.1% and cloning errors were eliminated from cloned alleles.Several properties of the data were examined to ascertain the utility of the genes for phylogenetic analysis (additional file 2). None of the mitochondrial genes exhibited stop codons when translated into amino acids. A test of homogeneity indicated that sequences had similar base frequencies across taxa (p values for all genes were above 0.99; data not shown). As expected, mtDNA sequences showed an anti-guanine bias, except for ND6, which showed an anti-cytosine bias and is the only one of the six genes encoded by the L strand; all other protein-coding mitochondrial genes are encoded by the H strand.The majority of variable sites in the mitochondrial datasets were third-position substitutions, as expected for protein-coding genes. The mitochondrial sequence dataset included more variation than the nuclear dataset, with a mean percentage of variable sites ranging from 4.8 to 6.2 for mitochondrial genes compared to 0.3 (RAG1, a protein coding region) to 1.8 for nuclear genes.We calculated the relative substitution rate between mitochondrial and nuclear data by dividing the net average mitochondrial distance and the net average nuclear distance between the Bahamas individuals and the rest of the C. flaveola phylogeny. The mean substitution rate was approximately 16 times slower for nuclear genes than for mitochondrial genes.Plots of the transition/transversion ratio against uncorrected distances indicated that nucleotide substitution in the mitochondrial genome was not saturated (data not shown). All plots were linear whether plotted by position or by gene. Thus, all nucleotides were employed in the ensuing phylogenetic analyses of C. flaveola.Phylogenetic analyses: outgroupsThe closest relatives to C. flaveola [10] chosen here as outgroups to root our phylogenies (i.e. Tiaris olivacea, Loxigilla portoricensis and Melanospiza richardsoni) were divergent by at least 11.2% from a mitochondrial perspective and at least 2% from a nuclear perspective.Phylogenetic analyses: congruence among tree topologies using different approaches and different genesPhylogenetic trees generated using the three approaches – MP, ML and Bayesian – were congruent for both mitochondrial and nuclear datasets. We also failed to reject homogeneity of phylogenetic trees based on each mitochondrial gene region independently (p = 0.3) or each nuclear gene region independently (p = 0.07). Therefore, for the subsequent phylogenetic analyses, we combined all six mitochondrial genes for the mitochondrial dataset and all three nuclear genes for the nuclear dataset. Concatenation of the different genes for nuclear data can be problematic as independent sorting of alleles in different parts of the genome and stochasticity of lineage sorting may lead to erroneous species trees [11,12]. However, our results should be unaffected by this issue because the nuclear and mitochondrial trees are congruent, with the exception of the mtDNA of Hispaniolan birds (Figures 2 and 3). The GTR+I+G model of evolution was selected using the AIC criterion in Mr ModelTest for both the mitochondrial and the nuclear datasets.Figure 2Coereba flaveola phylogenetic relationships based on the combined mitochondrial dataset (ATPase 8/6, cyt b, BCO1, ND2 and ND6 genes) inferred from a Bayesian analysis and rooted using three outgroup species (Tiaris olivacea, Loxigilla portoricensis and Melanospiza richardsoni). Values above the major branches represent the number of substitutions per site, estimated as the mean value of the branch in all samples of the Markov chain where the branch appears. Values below each major branch represent posterior probability values. Sample names are composed of a locality code (same as in additional file 1) and a sample number.Figure 3Coereba flaveola phylogenetic relationships based on the combined nuclear Bfib5, Rag1 and CHDZ genes, inferred from a Bayesian analysis and rooted using three outgroup species (Tiaris olivacea, Loxigilla portoricensis and Melanospiza richardosoni). Letters following each sample name (a or b) represent the two haplotypes for each sample.Phylogenetic analysis using mtDNAWhen describing the phylogeographic results, we use the following area name codes: LA = Lesser Antilles; PR = Puerto Rico; GA = Greater Antilles; SCA = South and Central America; BH/QR = Bahamas/Quintana Roo; DR = Dominican Republic (island of Hispaniola); JA/CY = Jamaica/Cayman Islands). A consensus phylogenetic tree based on a Bayesian analysis combining all six mitochondrial genes identified four major mitochondrial clades representing BH and QR, GA (except PR), LA (including PR), and SCA (Figure 2). Relationships were strongly supported with marginal posterior probability values ≥ 0.99 for all 15 major nodes.The continental (SCA) clade of bananaquits was sister to the PR and LA clades, which were imbedded within the GA (excluding PR) clades. This topology indicates derivation of the continental clade of bananaquits from within the GA. Within the PR/LA clade, sequences from PR had a sister relationship to the rest of the clade, and bananaquits on Grenada and Saint Vincent form a distinct phylogenetic group sister to populations in the northern LA. Within the SCA clade, samples from Central America (Panama and Belize) were grouped and distinct from samples from South America. Within South America, two groups were distinguished: birds from Trinidad and Venezuela and birds from Peru and Bolivia. A sample from Mexico (Biological Station of Los Tuxlas, Veracruz) sequenced with ND2 (M. Miller, pers. comm.) fell within the Central America group (data not shown) when added to our mitochondrial data.Phylogenetic analysis using nuclear DNABayesian analysis combining all three nuclear genes (Figure 3) distinguished four clades: (1) BH, (2) JA and CY, (3) DR together with isolated haplotypes from Montserrat, Barbados, Saint Lucia and PR, and (4) PR, LA and SCA. All three genes showed the affiliation of continental haplotypes with PR/LA haplotypes and the distinctiveness of DR and JA/CY haplotypes. Nevertheless each gene shows a specific pattern. For example, BFib5, the most variable gene, unites the four DR and seven LA/PR haplotypes, a group that is separated from the other sequences by five nucleotide changes. CHDZ, the least variable gene, separates with only one nucleotide change the JA/CY samples on one side and DR samples on the other side from the rest of the phylogeny (Figure 4).Figure 4Haplotype networks for the three nuclear genes (Bfib5, Rag1 and CHDZ) and the combined genes for Coereba flaveola. Branch lengths are proportional to the number of mutations and the size of each node is proportional to haplotype frequency. Each of the clades defined from the mitochondrial or nuclear consensus trees (Figures 2 and 3) is given a specific color.Mitochondrial versus nuclear phylogenetic analysesIn comparison with the mitochondrial tree, the tree based on nuclear data resolved the more recent divergences poorly. For example, the nuclear data grouped the continental samples with those from the LA. Nonetheless, the nuclear phylogeny was consistent with the mitochondrial tree in the basal branching pattern for C. flaveola within the West Indies. The distinctiveness of DR birds in the nuclear tree was surprising given their relative proximity to JA/CY birds in the mitochondrial tree. Because the discrepancy between mitochondrial and nuclear data suggests a distinctive phylogenetic history for the DR population, we sequenced eight additional DR individuals with both mitochondrial (ATPase) and nuclear (BFib5) genes. Those additional sequences confirmed that DR birds grouped with CY and JA birds from a mitochondrial perspective and formed a separate clade from the other populations from a nuclear perspective (data not shown).Estimates of population splitting timesThe likelihood-ratio test based on the six mitochondrial genes failed to reject the null hypothesis of rate constancy (-lnL = 9243.85 enforced tree and 9217.02 non enforced tree, χ2 = 53.66, df = 40, p = 0.073). Therefore, to estimate divergence times, we used linear mtDNA substitution rates of 1.5 and 3% Ma-1, which span the range of most calibrations (see \"Methods\" for justification [13,14]).The average genetic distance between BH/QR birds and all other populations was 0.0562 ± 0.0037, placing the ingroup root between 1.75 and 3.99 Mya. The average divergence between the GA and LA/PR/SCA groups was 0.0529 ± 0.0038, indicating that the split between those two clades occurred at approximately the same time, i.e., 1.75 to 3.99 Mya. The divergence between the LA/PR clade and the SCA clade was 0.0234 ± 0.0025, implying a split about 0.69 to 1.72 Mya. Finally, PR diverged from the LA approximately 0.34–0.93 Mya.DiscussionIn this paper, we have described phylogeographic relationships of bananaquit populations to determine the history of colonization within the Caribbean Basin. Additional analyses using coalescent models to infer population parameters require a different sampling regime (i.e., more individuals per population and more nuclear genes) and will be reported separately (Bellemain et al. unpublished).We provide a well-resolved phylogeny of the bananaquit in the West Indies, based on multiple genes. Aspects of the bananaquit phylogeny highlight points of general interest that are not commonly appreciated for birds. First, we infer that populations sampled in the Greater Antilles and Bahamas were the source of mainland colonization for C. flaveola, which is the reverse of the usual continent-to-island direction of colonization. Second, our study demonstrated the dynamic nature of the bananaquit distribution, resulting from several phases of overlapping expansions, especially through the Lesser Antilles. Third, a discrepancy between mitochondrial and nuclear haplotypes among bananaquits on Hispaniola (Dominican Republic samples) suggested the recent replacement of the mitochondrial genome by introgression from Jamaica.Mitochondrial versus nuclear data: relative coalescence time, sorting of ancestral polymorphism and inferences concerning phylogenetic historyDNA sequences of mitochondrial genes provide a single estimate of a species phylogeny based on one linkage group [15]. In our analysis of bananaquit phylogeny, we adopted a multi-gene approach based on three nuclear genes as well as DNA sequences for six mitochondrial genes. The more rapid coalescence of mtDNA represents an advantage as it is more likely to resolve nodes with short internode distances, and the probability of incomplete lineage sorting is reduced compared to nuclear DNA [16,17]. However, an evolutionary reconstruction based on multiple independent genes increases confidence that the true species tree has been recovered, and improves phylogenetic resolution by averaging out potentially misleading effects of ancestral polymorphisms [16,18,19]. Furthermore, analytical problems resulting from the stochastic nature of lineage sorting and nucleotide substitution are reduced with an increased sample of genes and nucleotides. Our aim, here, was to explore the evolutionary history of the bananaquit populations within the West Indies from the vantage points offered by the different genes independently and collectively, considering their different mutation and substitution rates, coalescence times, modes of inheritance and effective population sizes.We obtained a well-resolved phylogeny of C. flaveola based on analysis of the six mitochondrial genes. The substitution rate of bananaquit mtDNA was high relative to the observed rate of lineage splitting, as evidenced by abundant synapomorphies and complete lineage sorting (reciprocal monophyly) of the major clades. The data exhibited no saturation and approximated clock-like nucleotide substitution. Thus, we are confident that the tree presented in Figure 2 represents a strongly supported hypothesis of matrilineal relationship.In contrast, nuclear genes demonstrated considerably lower resolution regarding the phylogenetic relationships of bananaquit populations. The substitution rate of the nuclear genes was, on average, 16 times slower than the mitochondrial genes, and thus closely related clades strongly supported by the mitochondrial data (e.g., north central LA sister to Grenada/St. Vincent, and JA/CY sister to DR) were not distinguished by the nuclear data. Nonetheless, the allelic divergence at nuclear loci and the relative frequency of nuclear alleles across populations (Figure 4) clearly identifies the genetic distinctiveness of the bananaquit clades we have named \"Bahamas\", \"Jamaica\" and \"Puerto Rico\", and lends strong support to the hypothesis that these clades separated early in the diversification of Coereba.The slow nuclear sorting rate relative to population diversification resulted in many examples of incomplete lineage sorting compared to the mitochondrial perspective. Alternatively, the nuclear data could be interpreted as revealing abundant gene flow mediated by male bananaquits, biparental gene flow followed by localized episodes of selective sweeps acting on the mitochondrial genome, or drift associated with reduction in female population size. We suspect incomplete lineage sorting because deep in the bananaquit phylogeny both the nuclear and mitochondrial data consistently identify the same clades, with the single exception that individuals on Hispaniola (DR) have mitochondrial DNA closely related to JA/CY bananaquits, whereas the nuclear markers are allied with those on PR and the north central LA.Phylogenetic history of the Dominican Republic population on Hispaniola revealed by the discrepancy between nuclear and mitochondrial dataNuclear markers separate DR from JA/CY birds, whereas those populations are grouped in the same clade from a mitochondrial perspective. This surprising result was confirmed by sequencing eight additional samples from this population for nuclear (BFib5) and mitochondrial (ATPase) genes (data not shown). Recent introgression of JA/CY mitochondrial haplotypes into the DR population is the most likely explanation of conflicting positions of the DR samples in the nuclear and mitochondrial trees (Figures 2 and 3). Although JA/CY mtDNA completely replaced the local DR mtDNA, we found no evidence of introgression in the nuclear genome. The challenge is to understand the complete introgression of the JA mitotype against a predominately DR nuclear background.Mitochondrial genomes are particularly susceptible to introgression, although this phenomenon is not clearly understood (see [15] for a review). The ca. four-fold smaller effective population size of the mtDNA genome compared to the nuclear genome implies a higher probability for introgression by mtDNA. However, this outcome would be relatively unlikely if the number of JA immigrants was small relative to the population of resident DR bananaquits. Of course, if immigration from JA coincided with a population crash of Hispaniolan bananaquits, the likelihood of stochastic fixation of the JA mtDNA clade would have increased accordingly.Alternatively, mitochondrial introgression could have been favoured by selection, while the nuclear genomes of the two populations were incompatible. This type of selection-driven introgression has been demonstrated in Drosophila from mtDNA microinjection studies [20] and more recently in wild goats from a molecular phylogenetic study [21]. The authors of the latter study suggested that proto-Hemitragus mtDNA could have invaded the ancestral population of Capra because the former were better adapted to high altitudes. However, we cannot imagine differences in the selective environments of basal bananaquit populations that might have led to the selective advantage of the JA mitochondrion, considering that the major islands of the Greater Antilles have a similar range of environments.A third hypothesis is that sexual selection could account for the observation, much in the same manner as hypothesized for Dendroica occidentalis warblers along the west coast and islands of British Colombia [22]. In this case, hybrid JA/DR males would have a mating disadvantage, slowing the introgression of nuclear genes relative to the mitochrondrial genomes passed on only by the females. Each backcross generation would dilute the contribution of JA nuclear alleles, while JA mitotypes might become fixed by chance. However, unless the JA mitochondrion experienced a selective advantage, this scenario is likely only in the case of greatly reduced DR populations.Other biological processes that can create topological incongruence between gene trees include lineage sorting, paralogy, and lateral gene transfer [23]. These alternatives can easily be discounted in the case of DR bananaquits.First, because several independent nuclear gene sequences support the complete divergence of the nuclear genomes of JA/CY and DR birds, the combination of DR nuclear and JA/CY mitochondrial genomes is not likely the result of incomplete lineage sorting of an ancestral polymorphism in nuclear alleles. Although scenarios implying gene duplications and gene deletions could be inferred for one nuclear gene only, it is highly unlikely that the same scenario may have occurred independently and identically in the three unlinked nuclear genes (RAG-1, Bfib5 and CHDZ). Indeed, the absence of substantial divergence between JA/CY and DR mitochondrial genomes indicates recent derivation. Second, phylogenetic trees using paralogous sequences may be misinterpreted if one assumes that the nuclear sequences are orthologous. Orthologous genes derive from the same locus whereas paralogous genes derive from different loci that originated by gene duplication [24]. However, reconstructing an erroneous gene tree based on paralogy or gene duplication/deletion is highly improbable because it would imply one multiple-gene or genome-wide duplication event at the root of the tree, a panmictic DR/JA/CY population until recently (necessary to explain the similar mitochondrial genomes), and several recent nuclear gene deletions. Finally, lateral gene transfer other than by hybridization/introgression can happen when an organism incorporates genetic material from another distantly related organism, and this can create topological incongruence between gene trees. However, animals seem to be largely unaffected by this phenomenon [25] and it has never been reported for birds.The evolutionary history of bananaquitsThe mitochondrial phylogeny pictured in Figure 2 confirms the phylogeographic structure of bananaquits revealed by the earlier RFLP analysis [3]. Regional mtDNA haplotype clades displayed levels of sequence divergence typically in excess of 2%, with the largest distances exceeding 5% (e.g., BH versus other populations). The nuclear DNA data confirm the mtDNA-based hypothesis of early divergence amongst bananaquit populations today revealed by three highly differentiated regional assemblages: BH, JA/CY, and PR/LA/SCA.Bananaquit lineages representing the BH, JA and PR were probably isolated soon after the initial geographic spread of the group in the GA. Subsequent expansions from the BH and JA were geographically restricted. Bananaquits from the BH probably spread to the Yucatán Peninsula of Mexico early in the history of that clade based on the mtDNA sequence of a single bird collected from QR (note that bananaquits from the coast of Veracruz group with other Central American birds, and that the species does not occur elsewhere on the Yucatán Peninsula, except for islands off the coast of Quintana Roo [26]. The sister relationship of this QR individual and the BH sample could place the root of the bananaquit phylogeny on the continent, independently of the widespread and more recent SCA clade. A more likely interpretation is that the QR individual represents relict population of a separate invasion from the Greater Antilles now restricted primarily to Cozumel Island. This raises the intriguing possibility that it is a relict of a former divergent population of bananaquits on Cuba, which would have been a likely source of colonization from the GA to QR. It seems unlikely that bananaquits never existed on Cuba. Their current status has been defined as \"possible permanent resident\" on cays off the northern coast of Cuba (Ciego de Ávila and Camagüey Provinces) and on the island itself at Gibara (Holguín Province), but breeding has not been established [27]. To summarize, bananaquits from coastal Yucatan deserve additional study, but data in hand suggest that the matriline allied to the BH is narrowly restricted in comparison to the predominant mtDNA haplotype clade in Middle America.The lineage of bananaquits found today on JA/CY is also narrowly distributed from a joint nuclear and mtDNA perspective. Given the predominance of the JA/CY matriline in the DR, however, it is clear that at least female bananaquits have moved between the islands relatively recently. Thus, migration or range expansion of the JA clade group has been recent in comparison to that of the BH mtDNA haplotype clade.Nuclear and mitochondrial data indicate that most of the contemporary distribution of bananaquits results from the spread of birds whose descendents are now found in PR, LA and SCA. The geographic expansion of this clade is relatively recent in comparison to the original diversification of bananaquits, and the first split of this expanding lineage (PR clade) separated island from continental birds. Subsequent diversification on the mainland led to reciprocally monophyletic mtDNA haplotype clades, one predominant in South America and the other in Middle America. In the islands, PR populations became isolated from LA populations, and then bananaquits in the north central LA became separated from the populations on Grenada and St. Vincent.The pattern of evolutionary quiescence followed by geographic expansion is also repeated in the mtDNA record of bananaquits in the LA. This history was clearly revealed in our earlier research [3] based on a mtDNA RFLP analysis of a larger sample of birds per island, and demonstrated that bananaquits had recently expanded their distribution through the northern islands in the chain. However, we did not anticipate the northward expansion of the north-central Lesser Antilles bananaquit clade all the way to the British Virgin Islands. It is particularly noteworthy that bananaquits crossed the Anegada Gap, which serves as a significant biogeographic divide in the West Indies, to colonize islands that would have been connected to PR, or nearly so, during Pleistocene low sea level stands. This seems improbable if the British Virgin Islands had been occupied by the Puerto Rican population of bananaquits, and suggests that a catastrophic event might have caused the extirpation of populations in the northern LA and the British Virgin Islands. The alternative scenario of north-central LA bananaquits displacing PR like birds in the British Virgin Islands as a result of competitive superiority is lessened by the evident failure of this lineage to invade Puerto Rico. Our sampling in PR included coastal areas on the eastern end of the island close to the Virgin Islands.At the other extreme of the LA, Grenada and Saint Vincent have maintained their evolutionary separation from islands to the north, but no significant separation from one another as measured genetically or morphologically. Both islands harbor melanistic forms of the bananaquit, as well as shared mtDNA haplotypes, that are not found on islands to the north. The evolutionary connection between Grenada and St. Vincent might reflect their separation by a shallow bank of islands (the Grenadines) that would have connected the islands during Pleistocene low sea level stands [28]. This is not the case for other islands in the LA, which are mostly (excluding St. Kitts-Nevis and Antigua-Barbuda) separated one from the other by deep-water channels.Phylogenetic support for taxonomic distinctions among subspeciesOur molecular systematic appraisal of bananaquits permits us to assess phylogenetic support for taxonomic distinctions among the many named subspecies. Two subspecies groups were recognized by Paynter [7]: bahamensis (from the Bahamas) and flaveola (representing all other populations), but most populations in the West Indies have received specific or subspecific epithets at some time in the past, Bond [29] recognizing 16 subspecies. Our data confirm the genetic distinctiveness of the bahamensis birds; however, the flaveola group encompasses mtDNA haplotype clades that are nearly as divergent as flaveola and bahamensis groups. For example the JA/CY/DR clade is approximately 5% diverged in mitochondrial sequence from the remainder of the populations recognized as the flaveola subspecies by Paynter [7]. Additionally, subspecies such as portoricensis on Puerto Rico represent distinct evolutionary lineages. However, more often than not closely related or identical mtDNA haplotypes encompass several subspecies. For instance, the northern LA mtDNA haplotype clade includes four subspecies (sancti-thomae, bartholemica, martinicana, and dominicana), the two Panama bananaquits comprise different subspecies (columbiana and aterrina), and birds from Bolivia and Peru belong to three different subspecies (intermedia, dispar and alleni). Although finer genetic distinctions consistent with morphological differences between populations may exist, this study adds evidence that presently recognized bird subspecies often do not represent historically and phylogenetically equivalent evolutionary lineages (e.g., [30]).Revision of earlier phylogenetic and biogeographic history of the bananaquitOur analysis also allows us to review the phylogenetic and biogeographic history of the lineage. Bond [31] interpreted the distribution of C. flaveola in the West Indies as resulting from two invasions, one from South America spreading north through the LA and west to JA, and the other from Central America spreading north and east to the BH. Our initial mitochondrial RFLP-based survey of bananaquits [3] indicated a different, more complex history. However lacking samples from bananaquit populations on Dominican Republic and the Bahamas, as well as a suitable outgroup, we could not satisfactorily address Bond's [31] hypothesis of a continental origin for the species [3]. Results reported here confirm and extend the earlier molecular phylogenetic analyses.The sister-group relationship between SCA bananaquit populations rejects Bond's hypothesis, which predicted that the two continental regions should be most closely related to the island populations founded from South and Central America, respectively.Islands as the ancestral area of the bananaquit and \"reverse colonisation\" of the continentBecause species diversity on islands decreases with distance from continental source areas [32,33], biologists have assumed that colonization of islands or archipelagos is a one-way process, and that \"reverse colonization\" to the mainland against a diversity gradient rarely, if ever, occurs. Our study confirms previous indications that island species also can colonize continental areas. Although the depauperate biotas of remote islands and archipelagos have little chance of recolonizing continental landmasses, near islands, such as the West Indies, have larger biotas with fewer impediments to dispersal, considerably improving the probability of back migration, or onward migration. Colonization from the islands to the mainland has similarly been reported within the West Indies for several bird species (e.g. Icterus orioles, [34]; Myiarchus tyrant-flycatchers [35]; possibly Amazona parrots, [36]). Island to mainland colonization has also been demonstrated in monarch flycatchers from the Solomon Islands to New Guinea/Australia [37] (see [38] for a review).In the case of the bananaquit, we have provided both mitochondrial and nuclear evidence that permits strong inference that birds from the islands have colonized the mainland. The nesting of the South American to Panama samples within the West Indian clades and the deep paraphyly of bananaquits from the BH and JA clade with respect to all other bananaquits, including all mainland birds (except the single QR individual), represent the strongest lines of evidence that the ancestral node of the extant lineages is most parsimoniously placed in the Greater Antilles. Furthermore, ancestral area analysis (data not shown) based on the Bremer method [39] clearly excludes the continent as the ancestral area of Coereba. Finally, near outgroups for the bananaquit are principally emberizid finches from the West Indies [10]. We cannot place the origin (stem lineage) of the species itself, but this issue is not relevant to the present study.To conclude, islands, including the West Indies, might be significant sources of biodiversity for continents, and we emphasize the importance of considering reverse colonisation for interpreting biogeographic patterns.Diversification and range expansion of Coereba flaveola in the West Indies: alternative scenariosAlternative scenarios of diversification and range expansion, from the Greater Antilles/Bahamas, can explain the phylogenetic patterns observed in both the mitochondrial and nuclear gene trees. They differ mainly in whether the continent was invaded directly from the Greater Antilles or through the Lesser Antilles.In the simplest scenario, bananaquits expanded from the GA to independently colonize the LA (through PR) and the continent (directly from the GA) approximately 1.7 to 4 Mya, through an Eastern route. Considering the affinity of South American genotypes to those on PR rather than DR or JA, a Western route can be excluded. This scenario is consistent from a mitochondrial point of view. It does not exclude the possibility that several phases of expansion might have occurred within the LA (including a recent one that would have homogenized the gene pool across the northern Antilles). However, direct colonization of South America from GA seems unlikely for several reasons. First, bananaquits, which do not flock and are not strong fliers, would have difficulty making a long over-water flight. Second, virtually all colonization events in the West Indies occur in stepping-stone fashion from one island to the next, rather than over long distances [2,40]. Third, remnants of the nuclear genome of the South American birds in PR and LA (Figure 3) favour dispersal through the islands rather than directly from the GA.We envision colonization from GA to SCA through LA as occurring in the following way (Figure 5). First, bananaquits spread from the DR to PR and through the LA approximately 1.7 to 4 Mya. Evidence of this expansion is seen in the clade of haplotypes in the nuclear phylogeny recovered from DR, PR, and LA. This expansion did not reach the continent of South America.Figure 5Phylogeny of Coereba flaveola surimposed on the geography of the Caribbean region. The geographic patterns of the different mitochondrial and nuclear lineages allow resolving the complex phylogenetic history of the species.A second expansion would have occurred from PR through the LA to SCA about 0.7 to 1.7 Mya, as seen in the phylogenetic link between SCA, PR, and LA populations, exclusive of other GA populations, particularly DR (Figure 2). This is also corroborated by the fact that the combined nuclear genes (Figure 4) show the affiliation of continental haplotypes with LA and PR haplotypes, to the exclusion of DR haplotypes. It is also possible that the DR/PR/LA and PR/LA/SCA clades represent the same expansion, followed by lineage sorting.A third, more recent expansion from PR through the LA, but not to the continent, is indicated by the shared mitochondrial haplotypes of those two populations about 0.34 to 0.93 Mya. After the first and second expansions, PR birds would have had sufficient time to diverge from DR and continental populations from a mitochondrial perspective, but incompletely from a nuclear perspective. This would explain why the LA clade does not include DR mitochondrial sequences but does retain remnants of the DR nuclear genome.ConclusionAnalysis of genetic diversity in the bananaquit shows that the different rates of nuclear and mitochondrial genome processes, and incongruities between nuclear and mitochondrial phylogenies, can be used to dissect the often complex phylogeographic history of populations. We emphasize the importance of including multiple genes with distinct evolutionary histories. Our study is the first, to our knowledge, to demonstrate such biogeographic complexity for a single species. First, we demonstrated that the ancestral node of the extant bananaquit lineages is most parsimoniously placed in the Greater Antilles, indicating that islands, including the West Indies, might be significant sources of biodiversity for continents. We suggest that the \"reverse colonisation\" phenomenon deserves more systematic attention in phylogenetic analyses, and assessing its significance is of prime importance in studies of biogeography and ecology. Second, several phases of colonization and mixing among islands occurred before the bananaquit populations became isolated long enough to develop reciprocal monophyly in mtDNA. Third, the mixed nuclear and mitochondrial genomes in the Dominican Republic and the remnants of old Dominican Republic nuclear lineages in the Lesser Antilles suggest that expanding bananaquit populations mix with resident populations during expansion phases. Thus, genetic compatibility is maintained over periods exceeding three million years and perhaps much more, despite morphological and song differentiation. This does not exclude the possibility that expansions can follow extinctions of local populations or that colonists can competitively exclude local populations, as suggested by non-overlapping distributions of recently expanded mtDNA lineages in several species of Lesser Antillean birds [41].MethodsSamplingWe obtained tissue or blood samples from one to three C. flaveola individuals from 16 islands within the West Indies (Antigua, Bahamas, Barbados, Barbuda, British Virgin Islands, Cayman Islands, Dominica, Dominican Republic, Grenada, Guadeloupe, Jamaica, Martinique, Montserrat, Puerto Rico, Saint Lucia and Saint Vincent) and seven countries in Middle and South America (Belize, Bolivia, Mexico, Panama, Peru, Trinidad and Venezuela) (Figure 1). No voucher specimens were collected in the West Indies because identification of species is not an issue. The sampling locations cover the entire distribution of bananaquits within the West Indies, and are broadly representative of the geographic range of the species in Central and South America. Bananaquits occur only accidentally in Cuba and Florida [42]. To root the bananaquit phylogeny, we used the following outgroups: two individuals of Tiaris olivacea (from Puerto Rico, PRTOL and the Cayman Islands, CYTOL), two Loxigilla portoricensis (Puerto Rico, PRLPO1, PRLPO2), and one Melanospiza richardsoni (Saint Lucia, SLMRI), which are recognized as close relatives of C. Flaveola [10]. Altogether, our sampling included 49 individuals. Additional file 1 presents details for sample localities. Blood and tissue collection was non-destructive as described in Seutin et al. [3]. On Grenada and St. Vincent, we sampled bananaquits of the typical yellow form as well as the melanistic form [43-45].DNA extraction and sequencingTotal cellular DNA was extracted from most samples using the CTAB extraction procedure described in [3]. The following six mitochondrial genes were amplified: ATP synthase 8, ATP synthase 6 (ATPase 8, ATPase 6, 852 bp); cytochrome b (cyt b, 717 bp), cytochrome oxidase (COI, 651 bp), Nadh dehydrogenase 2 (ND2, 1023 bp), Nadh dehydrogenase 6 (ND6, 501 bp). In addition, we amplified three nuclear genes: the recombination activating gene-1 (RAG-1, 1009 bp [46]), the fifth intron of the beta fibrinogen gene (Bfib5, 545 bp [47]), and the chromo-helicase-DNA-binding protein, which is sex-linked (CHDZ, 495 bp [48]). In total, we sequenced 3744 nucleotides of mitochondrial genes and 2049 nucleotides of nuclear genes. Negative controls were included in all PCR amplifications to confirm the absence of contaminants. Amplification of PCR products was verified by visualization on 1.5% TBE agarose gels stained with ethidium bromide. PCR products were then cut from 1.5% TAE agarose gels and left to incubate for two hours at 46°C in presence of gelase.Sequencing reactions were conducted with the gel purified products and BigDye chemistry (Applied Biosystems, Foster City, CA, USA), and cleaned using Sephadex G50 purification columns (Sigma). Sequencing reaction products were run on an ABIPrism 3130xl automated sequencer (Applied Biosystems). DNA sequence fragments were edited and aligned using Sequencher software version 4.5.We cloned samples in which nuclear sequences exhibited more than one heterozygous nucleotide site (double peaks of approximately the same height present on both strands) or double peaks extending through a portion of the entire sequence due to allele length variants. Cloning was accomplished using the Promega cloning kit (pGEM-T Easy Vector System II). Ligated plasmids were used to transform Escherichia coli competent cells (JM109) selected for inserts by growth on LB plates with IPTG and X-Gal (5'-3'). Positive colonies were grown overnight in LB and up to eight colonies were isolated in 50 μL of purified water. PCR, cycle sequencing, and purifications were then performed as previously described. For some individuals we sequenced one colony per clone and used the resulting sequence to deduce the sequence of the second allele from the original sequence based on the PCR amplification of genomic DNA. Where the original sequence was not interpretable, we sequenced between three and six colonies per clone to obtain clean sequences for both alleles. Cloning errors were identified by comparing multiple sequences and their consensus, and the error rate was estimated by dividing the number of cloning errors by the number of nucleotides sequenced.Our preliminary data indicated discordant nuclear and mitochondrial trees for Dominican Republic bananaquits (see results), which warranted increased sampling of individuals and genes. Accordingly, we sequenced the mitochondrial ATPase and nuclear BFib5 genes for eight additional birds from diverse locations across the Dominican Republic (additional file 1)Phylogenetic analysesNucleotide composition, nucleotide bias, percentage of variable sites and parsimony informative sites for both mitochondrial and nuclear genes were analyzed using PAUP* and Sequencher 6.1 [49]. We assessed saturation in the mitochondrial dataset by plotting the transition/transversion ratio against uncorrected genetic distance for the combined mitochondrial dataset.Phylogenetic analyses for each mitochondrial gene independently, for each nuclear gene independently, and for combined datasets followed verification of the homogeneity of gene regions in combination. Congruence among tree topologies generated using the six mitochondrial genes combined or among the three nuclear genes combined was tested with the partition homogeneity test in PAUP*, with 1000 heuristic replications [50,51]. In turn, we used three approaches to infer relationships among bananaquits. Maximum parsimony (MP) analysis was performed using heuristic searches with TBR branch swapping and support for interior nodes was assessed using a bootstrap approach (1000 replicates). Maximum likelihood (ML) analyses used MrModelTest v2.2 [52] to select the best-fit nucleotide substitution model for the data. Bayesian phylogenetic analyses were conducted using MrBayes v.3.0b4 [53]. We estimated posterior probability distributions by allowing six incrementally heated Markov chains to proceed for two millions generations, with samples taken every 1000 generations. The Markov chain of interest was considered to have converged when stationarity was reached, which we determined by plotting the posterior probability values of nodes against generation time in a cumulative fashion. Burn-in samples were discarded from the two separate analyses, and the remaining samples were combined to create a consensus phylogeny and to estimate posterior probability values and branch lengths.After fixing the topology of the consensus Bayesian tree, we implemented a |2 test of log-likelihood ratios to compare the difference in support between trees with and without a molecular clock enforced [54] so that we could evaluate the constancy of nucleotide substitution rate within the combined mitochondrial gene dataset. We analyzed the nucleotide substitution rate only for bananaquit lineages and did not include the outgroups.We failed to reject the null hypothesis of rate constancy in our data. It was not possible to obtain a robust calibration for the application of the molecular clock, first because passerines are poorly represented in the fossil record, and are difficult to identify to species when they do occur, and secondly because calibration information such as radiometric ages of heterochronous sequences or inferred ages of lineage splitting events [13] are not available for the studied species. However, our aim was to provide an approximation (not precise dating) for times of lineage splitting. Therefore, we used a broad range of linear substitution rates of 1.5–3% My-1 reported for bird studies [13,55]. To estimate divergence times, we considered the net average genetic distance between pairs of clades and its standard deviation, using a Tamura-Nei model calculated using MEGA software, version 3.1 [56] and divided the mean genetic distance by 1.5–3%.We employed the ancestral area method from Bremer [39] to infer the direction and sequence of island and mainland colonization of C. flaveola in the West Indies. This is a cladistic method based only on the topology of the tree, which is used to infer a gain-loss ratio for each branch of a simplified area cladogram. The cladistic analysis was based on the topology of the Bayesian tree for the combined mitochondrial dataset.We used the software NETWORK 4.111 [57] to estimate gene genealogies for each of the nuclear genes and for the combined set of nuclear genes, and to construct unrooted minimum spanning networks. A partition homogeneity test in PAUP*, with 1000 heuristic replications, was used to test for incongruence among tree topologies generated using the seven nuclear genes.Authors' contributionsEvB carried out the molecular genetic studies, participated in the sequence alignment and phylogentic analyses and drafted the manuscript. ElB and RER conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.Supplementary MaterialAdditional file 1Table 1. Blood and tissue samples used in this study with sampling locations, geographical coordinates and Genbank accession numbers for each sequenced gene. For nuclear genes, each accession numbers corresponds to an allele sequence. NA stands for \"none available\" (a clean sequence could not be obtained), NS for \"not sequenced\", numbers beginning with a \" A\" refer to previously published sequences.Click here for fileAdditional file 2Table 2. 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+    "id": "PMC2533022",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2533022\nAUTHORS: Javier Gayán, Antonio González-Pérez, Fernando Bermudo, María Eugenia Sáez, Jose Luis Royo, Antonio Quintas, Jose Jorge Galan, Francisco Jesús Morón, Reposo Ramirez-Lorca, Luis Miguel Real, Agustín Ruiz\n\nABSTRACT:\nBackgroundThe difficulty in elucidating the genetic basis of complex diseases roots in the many factors that can affect the development of a disease. Some of these genetic effects may interact in complex ways, proving undetectable by current single-locus methodology.ResultsWe have developed an analysis tool called Hypothesis Free Clinical Cloning (HFCC) to search for genome-wide epistasis in a case-control design. HFCC combines a relatively fast computing algorithm for genome-wide epistasis detection, with the flexibility to test a variety of different epistatic models in multi-locus combinations. HFCC has good power to detect multi-locus interactions simulated under a variety of genetic models and noise conditions. Most importantly, HFCC can accomplish exhaustive genome-wide epistasis search with large datasets as demonstrated with a 400,000 SNP set typed on a cohort of Parkinson's disease patients and controls.ConclusionWith the current availability of genetic studies with large numbers of individuals and genetic markers, HFCC can have a great impact in the identification of epistatic effects that escape the standard single-locus association analyses.\n\nBODY:\nBackgroundMost common diseases have an etiology so complex that years of research have yielded scarce results towards the elucidation of their causes. Technology and methodology are improving quickly but results have been arriving slowly. Nonetheless, optimism is in the air, because large studies of many individuals and genetic markers seem to finally be revealing some of the genetic factors behind these common diseases [1].The difficulty of elucidating the genetic basis of complex diseases roots in the many factors that can affect the development of a disease. Many factors, both genetic and environmental, each with possibly only a small effect, may be necessary for the expression of a particular disease phenotype. For example, most associations reported in the recent wave of genome-wide association studies of different common diseases exhibited small (1.1–1.4) to moderate (1.5–2) odds ratios [2].These small effects may only be detectable by means of genetic association analysis in very large samples, or in smaller sub-samples in which, by sample selection, this effect is enlarged: a sub-sample where the allele frequency of a particular risk gene is increased; or a sub-sample where a combination of other alleles or environmental factors act to increase the observable effect of a particular gene [3].Many genes may contribute to the expression of complex diseases. It is quite reasonable to expect that the effects of some of these genes do not sum up in a simple fashion. Epistasis generally refers to an interaction between the effects of genes at different loci, although the term has been used in different contexts by different disciplines [4]. Some of these genetic effects may interact among them, such that the presence of two or more particular genes may increase the risk to a disease more than expected from their independent effects, the expectation being derived from a pre-defined model, such as additive or multiplicative. For example, the odds ratio for an epistatic effect of two genes may be larger, even much larger, than the combined effect (sum or product) of each of the two single genes [5,6]. Moreover, there are biological models of epistasis where genes only have epistatic effects [7], such as a two-locus mutation masking a known phenotype. Some of these genetic effects may prove undetectable by current single-locus methodology [8]. There have been some early attempts to search for epistatic effects [5,9-11], but there is currently a need for methods to study this important genetic phenomenon, perhaps key for complex diseases.A wealth of current research in molecular genetics has discovered millions of genetic markers which provide a good coverage of common genetic variation across the entire human genome [12]. At the same time, advances in genotyping technology have greatly increased the quantity and quality of genotypes. Current genotyping platforms can generate millions of genotypes in short periods of time. These events have made possible the genetic association analysis of a trait across the entire genome.Although the arrival of genome-wide association testing is great news for the genetic dissection of complex traits, the large number of statistical tests involved raises the issue of statistical significance. For example, to maintain a Type I error of 5 percent when testing 100,000 markers for genetic association may require a test-statistic with a probability value of 5 × 10-7, if a Bonferroni correction is applied. Nonetheless, many of these markers are correlated so this correction would be too strict, but in any case the required p-value would be very small.This problem of multiple testing is even more extreme for the test of epistasis. For example, for 100,000 markers, there are a total of 5 × 10+9 two-locus combinations, which would require a Bonferroni-corrected p-value of 1 × 10-11 for a genome-wide significance level of 0.05, which again would be overly conservative due to the correlated nature of many of these tests. To achieve these significance levels it is necessary to study large samples and expect to find large epistatic effects.Replication of findings in independent samples is sought for growing confidence in statistical results. The lack of replication of association results may be due to different causes, some technical (low power due to small samples, bad quality of phenotypic or genotypic data, uncorrected noise or covariates) and some biological (heterogeneity of effects or population-specific risks). An approach to tackle the multiple testing issue is to divide the available sample into independent groups and to carry out the analysis in these independent groups to look for consistent results across the groups. Some true genetic effects will be missed due to lack of power (due to the reduced sample in each group) and to heterogeneity, but this approach may allow the identification of moderate/large-sized epistatic effects that are frequent and consistent.In this scenario, we have developed an analysis tool to search for genome-wide epistasis in a case-control design. Hypothesis Free Clinical Cloning (HFCC) is an standalone software which allows for single-locus genetic association testing, as well as epistasis testing for multi-locus combinations of markers. Due to the intense computational burden, it is programmed to take advantage of computer clusters by dividing the tasks into processes which can migrate to the available CPUs. We present here the method, as well as a genome-wide two-locus epistatic analysis performed on a real dataset of Parkinson's disease that illustrates the method. With the current availability of genetic studies with large numbers of individuals and genetic markers, HFCC can have a great impact in the identification of epistatic effects that escape the standard single-locus association analyses.MethodsInput DataSampleThe standard input to HFCC is a case-control sample with hundreds or thousands of individuals. Similarly to other association methods, it is convenient that cases and controls are matched for potentially important covariates like age, sex, ethnicity, geographical location, environmental factors, etc. Dichotomous phenotypes with a potential effect on the trait may be used as covariates in the analysis.If all cases have only one disease phenotype or trait, HFCC carries out a single-phenotype analysis. In the single-phenotype scenario, HFCC can analyze the full sample simultaneously for extra statistical power. In addition, we have developed a multi-group analysis strategy, explained in more detail in a later section, that allows the replication of consistent results, and it also aids the elimination of false positives results, a very attractive quality for genome-wide analysis of large number of genetic markers. For this single-phenotype multi-group analysis option, cases can be sub-divided into groups to allow for replication of consistent results across these groups. Controls can also be sub-divided into groups to eliminate spurious associations.Nonetheless, one of the strengths of HFCC is that it can analyze multiple phenotypes simultaneously. For this multiple-trait analysis, several groups of cases with different but related phenotypes are formed, each matched to its own control group. Indeed, the multi-group analysis strategy is especially convenient for the simultaneous analysis of several related diseases, or different symptoms of a syndrome, so that we can identify the genetic effects common to the different groups.GenotypesHFCC can currently analyze di-allelic markers such as single nucleotide polymorphisms (SNP), and it can also accommodate other dichotomous markers such as the presence or absence of a particular allele of a multi-allelic marker or of a haplotype. HFCC can analyze anything from small sets of candidate gene markers to genome-wide arrays of hundreds of thousands of markers.Markers can be filtered out before analysis if they have low call rate, low minor allele frequency, or if they fail a Hardy-Weinberg equilibrium test. Nonetheless, data analysis filters inherent in the software can eliminate, at least partly, these problematic markers.Currently, linkage disequilibrium (LD) among markers is ignored during the analysis, although it is useful for the validation and interpretation of results.DatasetsTo illustrate the method we used data from the SNP Resource at the NINDS Human Genetics Resource Center DNA and Cell Line Repository . The original genotyping was performed in the laboratory of Drs. Singleton and Hardy (NIA, LNG), Bethesda, MD USA [13]. We have used data on 270 patients with Parkinson's disease and 271 normal control individuals who were genotyped for 396,591 SNPs in all 22 autosomal chromosomes using the Illumina Infinium I and Infinium II assays. Cases were all unrelated white individuals with idiopathic Parkinson's disease and age of onset between 55–84 years (except for 3 young-onset individuals). The control sample was composed of neurologically normal, unrelated, white individuals from the USA.To explore the power of HFCC we also analyzed a simulated dataset that was originally generated to evaluate the power of a different gene-gene interaction method [14]. These case-control data were simulated under different genetic models, and different sources of noise (genotyping error, missing data, phenocopy and genetic heterogeneity). For each genetic model and noise condition, 100 datasets were generated. Each dataset contains 200 cases and 200 controls, with genotypes for 10 SNPs under HWE. An epistatic effect with no single-locus marginal effect was simulated for a pair of SNPs, and the remaining 8 SNPs were simulated under the null hypothesis of no genetic effect. For the genetic heterogeneity case, two epistatic effects (each due to a different pair of SNPs) were simulated. More detail of these simulated datasets can be found in the original publication [14].HFCC ModellingStatistical testsSingle-Locus Association testsHFCC can perform a single-locus genome-wide association scan. A di-allelic marker with alleles A and B, has 3 possible genotypes: AA, AB and BB. For each genetic marker, HFCC performs three statistical tests, comparing each genotype against the other two. For example, the frequency of the AA genotype is compared against the combined frequency of the AB-BB genotypes, in cases and controls. This could be considered a dominance model test for the B allele. Similarly, recessive (BB versus AA-AB) and heterozygote models (AB versus AA-BB) are also considered. The test for association between the established genotypic classes and the case-control groups can be a Wald test or a chi-squared test with one degree of freedom (df).Multi-Locus and Epistatic Association testsHFCC can also perform a multi-locus genome-wide association scan. For a two-locus scan, HFCC first forms all possible combinations of two markers from all available markers. For each two-marker combination (marker 1 with alleles A and B, and marker 2 with alleles C and D), there are 9 possible genotypic classes (AACC, AACD, AADD, ABCC, ...., BBDD), and a total of 512 fully penetrant disease models [7]. For our purpose, this number of models can be reduced to 255, because some models are redundant or represent a model with no genetic effect. These 255 models include a variety of standard models (single-locus, double-recessive, double-heterozygote, etc.) as well as other rare models. The user can select to test all available models or a subset of them.As in the single-locus case, the statistical test involves comparing the frequency of two sets of genotypic classes in cases versus controls. The two sets of genotypic classes are defined by the model being tested. For example, in the case of the double-recessive model, the frequency of the BBDD genotypic class is compared against the combined frequency of all other genotypes (AACC through BBCD), in cases and controls, from which a Wald-test Z statistic, or a chi-squared statistic with one degree of freedom, can be obtained.Another analysis option is a more general multi-locus test [6] which compares two chi-squared statistics with four degrees of freedom (one obtained for cases and the other one for controls). This test, although more general, may not be able to detect some disease models.For a three-locus scan, there are 27 possible genotypic classes, and the software is currently implemented to test the 27 simplest 3-locus genetic models, comparing the frequency of a genotypic class (ie, AACCEE) against the combined frequency of all other genotypes.Post-Hoc testsA variety of post-hoc statistical tests are included in the post-hoc analysis software, named Alambique. For single-locus analysis, it is possible to carry out Hardy-Weinberg equilibrium, heterozygous, homozygous, allele positivity (dominance), recessive, common odds ratio (Armitage's trend) [15], and genotypic (2 degrees of freedom) tests.For the two-locus analysis, all 255 fully-penetrant models can be tested. Moreover, interaction indices can be estimated to determine to which extent an observed two-locus association deviates from an additive model [16]. Finally, departures from a multiplicative model can be tested using a case-only chi-squared test [17].These post-hoc analyses are useful to determine which genetic model explains better an observed association, and to measure the epistatic component of associated multi-locus combinations.GroupingReplication (Case versus control) groupsThe statistical tests for association of a trait with a marker are based on the differential frequency between cases and controls of a particular allelic or genotypic combination. Comparing the full sample of cases versus the full sample of controls is the most powerful approach to find statistically significant associations. Nonetheless, to evaluate genomewide association or epistasis we need to test a very large number of markers or marker combinations, which in turn can produce a large number of spurious results. Therefore, we need to improve the filtering of false-positive results, at the expense of increasing the false-negative findings.We have approached this multiple-testing issue by partitioning the available sample into multiple replication groups. Cases are selected randomly to be part of one and only one case group, and similarly for control individuals and control groups. Then, the statistical analysis is performed on each of the paired case-control groups, and results that are consistent across all replication groups are selected. This sample-splitting technique may prove less powerful to detect positive results in general, but provides a powerful tool to eliminate false positives, thereby highlighting potentially true effects that are consistent across samples. The number of groups into which the sample is partitioned can be chosen by the user depending on the study design, and the number of subjects and markers available for analysis.This multi-group strategy is one of the differentiating aspects between HFCC and conventional association methods, and it is particularly useful for the analysis of multiple related phenotypes. Case groups are directly defined by phenotypic criteria, and control groups are matched to each case group. This type of analysis can reveal genetic associations that are common to these phenotypes, revealing a common etiology for multiple symptoms of a disease, or for several related diseases [18].Control filter (Control versus control) groupsHFCC has developed an efficient noise filter, by applying the association tests to independent groups of controls. Positive results that arise from a comparison of two groups of controls must be spurious associations due to marker or sample characteristics. These spurious associations can be filtered out of the results from the case-control analysis, providing an efficient sample-specific background noise filter.Algorithm and program executionHFCC requires specific data and parameter files. The input data file is a simple-text matrix of integers in which rows represent genetic markers and columns represent individuals. Each integer represents the genotype for one individual at one marker, coded as 0 for missing, 1 for one homozygote, 2 for the heterozygote, and 3 for the other homozygote. These integers are entered sequentially, with no blank spaces or lines, to save space. There is one data matrix for each group of cases (F1.txt, F2.txt, etc.), controls (C1.txt, C2.txt, etc) and noise-filter controls (CF1.txt, CF2.txt, etc.), as can be seen in Figure 1. A parameter data file defines the type of analysis (single- or multi-locus; genetic model; test statistic), number of genetic markers, number of groups, sample size per group, statistical cut-off for significance, and other necessary parameters.Figure 1HFCC data groups and algorithm. HFCC data groups are divided into cases (F), controls (C) and control filter (CF). The number of replication groups is experiment-specific and depends on the available dataset (sample size and number of genetic markers). The HFCC algorithm is defined by a particular analysis flow. For each marker variable (single-locus markers, two-locus marker combinations, etc. depending on the type of study), a sequential number of tests is performed. Case-controls comparisons are performed on each replication group, and control-control comparisons are carried out in each control filter group. If any of these tests is not beyond a statistical threshold, the marker variable is dropped, and the next marker variable is analyzed. Marker variables over the statistical threshold in all case-control tests, and below the threshold in all control-control tests, are selected.The following algorithm describes HFCC analysis flow (Figure 1). Genetic markers are analyzed sequentially in the order in which they are entered in the data matrices. First, HFCC selects a marker variable (either a single-locus marker or a multi-locus combination of markers, depending on the type of analysis). Then, for the selected analysis model, it computes genotype frequencies and test statistics in each replication group, sequentially. If the test statistic is smaller than a pre-defined cut-off in any of the replication groups, the marker variable is dropped at that stage and no more computations are performed on this marker variable to save processing time. If the test statistic is larger than the cut-off in all replication groups, then the marker variable is written to the output file and also entered in the control noise filter. This noise reduction analysis computes frequencies and test statistics in each control filter (control versus control) group sequentially. If the test statistic is larger than a pre-defined cut-off in any of the filter groups, the marker variable is flagged for removal from the final output file. The marker can fail at any of the sequential group analyses (replication or control filter group comparisons), at which point that marker is dropped to save processing time. This procedure is repeated for all possible marker variables, writing to the output file all associations considered statistically significant. This output file contains the marker (or marker combination) and the genetic model for which it yielded a positive association (ie, for a two-locus combination: marker 1, genotypic class 1, marker 2, genotypic class 2).These selected marker variables can then be included in two sequential post-hoc analyses which aid in the interpretation of the results. The first post-hoc analysis yields odds ratios and chi-squared values for all selected marker variables. It also applies a direction filter, by selecting only those results which display the same direction of effect in all replication groups, and writes a new output file, which contains only those marker variables with a significant and consistent effect (same direction and same model) in all replication groups.The second post-hoc analysis (Alambique) identifies the best type of genetic model for each association selected in the first post-hoc analysis. For this reason, all replication groups are combined into a single group, that is, all cases in one case group and all controls in a control group. Alambique has its own noise elimination algorithm, denominated tracking filter. Many of the selected associations are due to what we call a tracking marker, a locus with a very large marginal effect. Many of these tracking markers are not in HWE and are filtered at this stage. In any case, having a large marginal effect makes a locus appear in many two-locus associations, because the large effect tracks other loci. Because our focus is on finding epistatic interactions, the many positive associations due to these tracking effects can be filtered out and analyzed independently. For the remaining marker variables, which have passed this tracking filter, there is one analysis option that groups the two-locus results into those with marginal effects for only one locus (conditional effect), or for both loci (simultaneous effect) or those without marginal effects (epistatic effect) [19]. Some of the conditional and simultaneous results may still exhibit epistatic effects if the two-locus effect size deviates from an additive or multiplicative expectation, and these epistatic effects can also be flagged. A second analysis option tests all 255 fully-penetrant models in selected marker variables, to help choosing the model that best fit the data.Due to the extremely time-consuming and computer-intensive nature of these analyses, especially for genomewide multi-locus analysis, it will often be necessary to run HFCC under a small selection of genetic models, and then use Alambique to identify the best genetic model for the selected markers. The number and selection of genetic models to analyze will depend on the phenotype, the dataset, previous knowledge and computer resources available.ResultsWe have applied HFCC to both simulated and real datasets to illustrate the power and the functioning of the method.Simulated data analysisTo evaluate the power of the method, we analyzed a simulated dataset published previously [14]. Data were simulated under three types of two-locus epistasis models: Model 1 was a logical XOR model, a combination of exactly one heterozygous and one homozygous loci (i.e., for two markers with alleles A, B and C, D respectively, risk genotypes would be ABCC, ABDD, AACD and BBCD), similar to M170 [7] but with variable penetrances; Model 2 involved the inheritance of exactly two, and only two, risk alleles (i.e., A and C) from any of two different loci (i.e., risk genotypes would be AADD, ABCD, and BBCC), such as M84 [7]; Model 3 was a variable penetrance risk model with a protective double-heterozygote (i.e., protective genotype would be ABCD), similar to M16 [7]. In addition, to make the simulated data more similar to real data, and therefore to evaluate the effect of noise in the detection of these epistatic effects, different sources of noise were modeled in the simulations. For each of the three types of genetic models described above, the data was simulated and analyzed under six types of noise: without noise, with 5% genotyping error (GE), with 5% missing data (MS), with 50% phenocopy (PC), with 50% genetic heterogeneity (GH), and with all sources of noise simultaneously. For more information on these simulated datasets please refer to the original publication [14]. Power was defined as the proportion of times the correct model was detected out of each set of 100 simulations. For genetic heterogeneity the correct model was defined as detection of either one of the two epistatic loci simulated.To detect potential epistatic loci, we ran HFCC two-locus analysis with nine simple genetic models, the M1, M2 and M16 models [7]. There are a total of 45 possible two-locus combinations that can be formed with the 10 simulated genetic markers. Due to this relatively small number of tests (relative to a genome-wide study), HFCC was performed with only one group of cases and controls, and a chi-square cut-off of 6.64 (approximately a p-value = 0.01 with 1 degree of freedom). Using only one case-control group allows also for direct comparison of HFCC's power to the power estimates published previously for this dataset. Results from the SNPs simulated under the null hypothesis reveal fewer Type I errors than expected, confirming that the method is not biased and perhaps conservative. This reduced number of false positives may reflect that some of the epistasis tests used were correlated, as well as a general lack of power to detect some epistatic effects, especially for rare genotypes.HFCC had excellent power (>96%), with a Type I error of 0.01, to detect the simulated two-locus interactions under most genetic models and noise conditions, including genotyping error and missing data (See Table 1). Genetic heterogeneity reduced the power to 82% under model 3, and the presence of phenocopies had also a significant impact on power for models 1, and especially, 3. Finally, if all four sources of noise were acting simultaneously the power was reduced to 51% for model 1, 71% for model 2, and 34% to model 3.Table 1Power Analysis.Power (%)NoiseModel 1Model 2Model 3None10010099GE10010096MS10010097PC8910049GH10010082GE+GH+PC+MS517134Power of HFCC to detect a two-locus interaction at an alpha level of 0.01 under 3 genetic epistasis models and different sources of noise: no noise (None), genotyping error (GE), missing data (MS), phenocopy (PC), genetic heterogeneity (GH), and all sources of noise simultenously (GE+GH+PC+MS). For the datasets with genetic heterogeneity, we reported the power to detect either of the two simulated two-locus effects. Model 1 is a heterozygote-homozygote risk (i.e., risk genotypes ABCC, ABDD, AACD and BBCD); Model 2 represents a \"2 and only 2\"-allele risk (i.e., A and C) from any of two loci (i.e., risk genotypes AADD, ABCD, and BBCC); Model 3 represents a protective double-heterozygote (i.e., protective genotype ABCD).Parkinson's disease analysisTo demonstrate the HFCC software in real data we analyzed the open-access Parkinson's dataset described in the Methods section. We included all 396,591 SNP markers. Many of them had low minor allele frequencies or failed Hardy-Weinberg equilibrium (HWE), but were included in order to test the data filters inherent in HFCC, which are meant to eliminate, at least partially, these problematic markers.The 270 cases and 271 controls were separated into groups, to illustrate HFCC multi-group analysis strategy. We created 3 replication groups of 90 cases each (groups F in Figure 1) and 3 groups of controls of approximately similar size (C in Figure 1; one group with 91 individuals and two groups of 90 individuals). Because of the lack of more control individuals, the noise filter control groups (CF in Figure 1) were selected respectively from each of the control groups, chosen so that the noise filter would not pair two identical control groups (ie, CF1 = C3; CF2 = C1; and CF3 = C2). Ideally, independent groups of C and CF controls would be used, so we expected that the noise filter in this experiment, in which the same groups of controls were used as C and CF, would not be as effective.To detect potential epistatic loci, we ran HFCC two-locus analysis with nine simple genetic models, the M1, M2 and M16 models [7]. These nine genetic models were tested in all possible (78.6 × 10+9) two-locus marker combinations. In order to be considered a preliminary positive result, the chi-squared (1 df) cut-off value was set at 6.64, which yields a probability value of 0.01 for each replication group (p < 10-6 over all three replication groups). Although this p-value may be considered low compared to the number of statistical tests performed, this is only a cut-off to select preliminary positive results, which are then filtered and subjected to post-hoc analysis to select the most promising results. We obtained a total of 418,535 preliminary two-locus associations at this p-value cut-off (Table 2). To evaluate the impact of the different noise filters (control, direction and tracking) we applied them selectively to these preliminary results.Table 2HFCC analysis: Preliminary results and effect of noise filters.Applied FiltersUnfiltered ResultsAfter Control FilterAfter Direction FilterAfter Tracking FilterSelected two-locus SNP pairsSimultaneousConditionalEpistaticMethod I N418,535-320,265-71,332248,89835 %100%-76.5%-17.0%59.5%0.01%Method II N418,535-320,26526,37119,6306,70635 %100.0%-76.5%6.3%6.1%2.1%0.01%Method III N418,535340,043320,18826,34719,6116,70135 %100.0%81.2%76.5%6.3%6.1%2.1%0.01%Method I: Only Direction Filter applied.Method II: Direction and Tracking filters applied.Method III: All filters applied (Control, Direction, and Tracking filters sequentially in that order).N: Number of selected two-locus SNP pairs.%: Percentage of selected two-locus SNP pairs over the preliminary set (unfiltered HFCC results).A total of 36 SNPs were identified as tracking SNPs in the two methods using tracking filter.Noise filters (control, direction, tracking)Direction filterWhen the control filter is not used, the direction filter has a large impact on the number of results selected. Of the 418,535 preliminary associations, only 76.5 % (320,265) had effects in the same direction in all replication groups. These results can then be grouped into those with marginal effects in both loci (simultaneous), only one marginal effect (conditional), and no marginal effect (epistatic). A marginal effect was defined as a single-locus effect with a chi-squared (1 df) statistic larger than 3.84 (p-value < .05). This liberal cut-off serves our goal of selecting as pure epistatic effects those marker combinations with no or small marginal effects. Under these criteria, 22.3% (71,332) of the results had simultaneous marginal effects, 77.7% (248,898) had conditional effects, and only 0.01% (35) had epistatic effects. At this point, out of the hundreds of thousands of preliminary results, only 35 two-locus associations without marginal effects remained, very likely to be epistatic interactions. The many thousand simultaneous and conditional results may also involve epistatic effects on top of the marginal effects, but the large number of them prevents a thorough post-hoc analysis.Direction and Tracking filtersIn order to distil further the post-direction-filter results we can apply the tracking filter. Many of these two-locus associations are due to what we have denominated tracking loci, that is, markers with large marginal effects, which therefore display significant two-locus effects with many other markers. Most of these tracking markers exhibit large association effects because they fail HWE, and they are filtered out at this stage. In this Parkinson dataset we detected 36 tracking markers, defined as those markers showing up in 270 or more two-locus associations (top 0.1% of post-direction-filter results). Most of these tracking markers (83.3%) failed HWE (p < 0.001 in controls or <0.0001 in cases), and others failed minor allele frequency (MAF<0.1) or call rate (CR<90%) criteria. Therefore, results involving these markers can be safely excluded.A few of these tracking markers represent the best single-locus association results which, due to their large marginal effects, also appear in a large number of two-locus association results. These large-single-locus-effect tracking markers need their own specific post-hoc analysis. Because of their large marginal effects, these markers are likely identifiable by single-locus analysis, but it is still noteworthy to discover if they have epistatic effects, and with which genes. However, it is hard to identify a significant epistatic interaction in the background of such a large marginal effect. Individual post-hoc analysis for each of these markers may identify the most likely multi-locus combination involving those marker.Interestingly, by filtering out the marker combinations involving the tracking markers, we can eliminate 91.8% of the previous positive results. Most of these results were conditional effects, involving the tracking locus (with a large single-locus effect) and a tracked locus (with no main effect, but tracked by the large effect in the other locus). The post-tracking-filter results include a total of 35 marker variables with epistatic effects, 6,706 with conditional effects, and 19,630 with simultaneous effects.Control, Direction and Tracking filtersWe can repeat this data filtering process, but this time starting by applying the control noise filter. The control filter was set to employ the same statistical cut-off (chi-squared = 6.64) used in the case-control comparisons. Association results above this cut-off value obtained in analysis of two control groups can be deemed problematic and excluded from further analysis. This control filter was able to eliminate 18.75% of the preliminary results, leaving a total of 340,043 associations (81.25%). Applying the direction filter to the post-control-filter results eliminates only a further 5.84% of preliminary results, yielding a total of 320,188 associations, almost exactly the same quantity as when the direction filter was used alone. Therefore, the control and direction filters seem to eliminate most of the same results, which reassures their correct functioning and also allow for different analysis strategies. On one hand, when enough control subjects are not available and consequently the control filter can not be used, these results suggest that the direction filter may work well alone. On the other hand, the direction filter may not be useful for some multiple-phenotype studies, expecting effects on different directions in different replication groups. Thus, in some scenarios, the control and tracking filters may provide enough noise elimination by themselves.When the tracking filter was applied to the results selected by the control and direction filters, we obtained 35 two-locus combinations with epistatic effects, 6,701 with conditional effects, and 19,611 with simultaneous effects. By applying three consecutive data filters (control, direction and tracking), we excluded almost 94% of the preliminary results. The remaining 6.3% of results are subsequently analyzed for departures of additive or multiplicative two-locus models, to estimate their potential for epistatic effects.Epistatic interaction indicesHFCC analysis yielded 35 two-locus combinations with epistatic effects and no noticeable single-locus marginal effects, ten of which are displayed in Table 3. In addition, there are 26,312 conditional or simultaneous two-locus combinations, which may display interaction effects over and above the marginal effects. There are a variety of tests and indices to detect departures from additive or multiplicative models. For example, a case-only chi-squared test can detect two-locus interactions which deviate from a multiplicative model. For the present study, this case-only statistic was used to choose, among the conditional and simultaneous marker variables, those with the most significant interactions (Table 3). In addition, marker quality control criteria were checked to assure all markers in selected combinations passed a minimum requirement (HWE p-value > 0.001 in controls and >0.0001 in cases; MAF>0.1; call rate>90%).Table 3Selected Parkinson's disease two-locus combinations.SNP1SNP2EffectGenetic ModelOdds Ratio2-locus X2Case-only X2rs6542522rs3923511ETC*GA0.2824.9713.07rs10799573rs1341622ECC*CT5.2223.0810.61rs12520264rs2992630EAG*GA0.2223.8623.12rs324454rs12672177EGG*AG0.2124.3111.55rs6656554rs3898966EGT*TC3.8323.4422.34rs7650598rs12353255EAG*AG3.9923.949.40rs2439525rs1327918ETT*CC2.3922.047.13rs10201616rs4495512ECT*TT5.0424.088.63rs11167062rs1270919ETC*GT0.2021.8320.65rs2419117rs10512174EGT*CC5.5523.339.33rs1370699rs1673130CCC*CC0.3525.5030.15rs1590957rs8043401CTT*AG0.3224.1728.18rs1557615rs2582597CTC*AG0.3521.7626.38rs11781101rs4775501CCC*CC0.2223.9925.69rs1370699rs7897163CCC*CC0.3430.6525.68rs13197142rs2206699CGG*AA2.3321.8525.16rs732594rs12373417CAA*GG3.6222.3125.16rs10498269rs1551355CTC*CC0.3823.4225.00rs2555614rs331617CAA*TT0.3524.1524.22rs3894377rs10774863CAG*CT0.3125.4723.84rs4799327rs2301661SAG*CC4.3220.6533.62rs2336865rs7973385SGA*TC3.2230.7127.77rs6779648rs270406SGG*AG0.2626.5722.49rs12599027rs767055STT*TT0.2326.2324.34rs3891371rs4724620SAG*CC0.2935.7623.92rs2297518rs660454SAG*TT0.1223.9223.72rs357968rs1159145SAA*AA0.2630.2923.47rs1476097rs5766305STT*TT0.3723.9323.19rs2955005rs2169793SGT*AG3.2925.7422.76rs2560790rs9390939SAA*CA0.3223.3522.62A selection of two-locus marker combinations consistently and significantly associated to Parkinson's disease (p < 10-6) and displaying an epistatic (E) effect, or conditional (C) or simultaneous (S) effects with significant deviations of a multiplicative model. The genetic model tested is specified as SNP1 genotype * SNP2 genotype.Validation analysisIn order to validate to some extent these results, and without immediate access to a truly independent sample, we created 10 validation samples by randomly re-creating the case groups (F) from the full pool of cases (N = 270), and similarly for the control groups (C). In the described analysis of the Parkinson dataset, three replication groups were created randomly, to find consistent results across these groups. To examine the possible effect of this random group membership, the replication groups were re-created randomly and reanalysed in what we called validation experiments. These validation experiments are not permutation simulations to estimate the empirical significance level of parameter estimates, and are not a standard bootstrapping technique because they use sampling without replacement. The replication groups in each validation analysis were re-created under association (keeping cases as cases and controls as controls), and not under the null hypothesis of no association. These validation experiments are used to examine the consistency of results when the replication groups are re-created.The control noise filter was not employed for these validations analyses to save computing time, since it was found that for this dataset the direction and tracking filters alone were sufficiently efficient. Because we are using the same set of cases and controls in all the validation groups, a high degree of consistency of results across validations analyses would be expected a priori. Nonetheless, due to the small sample size of each group, the many requirements for the selection of marker variables, such as the consistency of strength and direction of result in three replication groups, and the subjective categorization (single-locus X2 > 3.84) of results into different types of effects (epistatic, conditional and simultaneous), a large variability in results across validation analyses is reasonable.The total number of preliminary results varies noticeably across validation groups (Table 4). This variability illustrates the randomness inherent in this type of analysis. The total number of two-locus combinations selected as simultaneous or conditional are much more consistent. Nonetheless, the number of epistatic effects is quite variable again, perhaps a consequence of the small number of marker combinations selected under this category.Table 4Validation analysis.SamplePreliminary ResultsSimultaneousConditionalEpistaticOriginal418,53519,6306,70635Validation 1396,70820,8556,89723Validation 2283,58419,2706,21918Validation 3299,84619,2616,00027Validation 4403,47519,6406,66021Validation 5337,42219,5756,46322Validation 6320,40618,8796,23020Validation 7313,65318,6675,94321Validation 8294,00719,2556,36026Validation 9322,75418,4785,77222Validation 10363,19419,3175,52928Average Count (St. Dev.)341,235 (47,125)19,348 (629)6,253 (419)24 (5)Average %100%7.92%2.56%0.010%Number of total preliminary results, and number of selected simultaneous, conditional and epistatic two-locus combinations in the original sample, and in ten random-placement validation samples. Average numbers (and standard deviations) for the 11 samples, and average percentage of each category, are included.In addition to the variability in the number of results, it is important to note the general lack of consistent results across validation groups. The simultaneous effects were the most consistent type of result. One two-locus combination (rs7653784 rs499091) showed up in the original analysis and the 10 validation samples. Five other combinations with simultaneous effects were consistent in 10 out of the 11 samples, and 28 combinations were consistent in 9 samples. Of the conditional results, thirteen marker combinations were consistent in 8 samples. Perhaps because their effect is harder to replicate, only one epistatic marker combination was present in as much as 5 samples, while 2 combinations were present in 4 samples, and three combinations in three samples.DiscussionHFCC is a new computer software for exhaustive genome-wide analysis of multi-locus association effects in a case-control design. It carries out different types of statistical tests to assess a variety of genetic and epistatic models. HFCC differs from other association or multi-locus methods in that it can analyze simultaneously multiple samples or multiple phenotypes, and incorporates several complementary noise-signal filters, and also post-hoc analysis tools. To address the enormous computing task, it is elegantly designed to take advantage of the multiple CPUs available nowadays in computer servers or clusters.The goal of HFCC analysis is to find multi-locus marker combinations which are significantly associated with a phenotype, especially those displaying interaction effects which may not be detected in a single-locus analysis. By setting the type of genetic model, the number of subjects in each group, the number of replication groups, the statistical cut-offs for the different tests, and applying different noise elimination filters, HFCC can arrive at a selection of the most promising multi-locus combinations.Other multi-locus methods to detect gene-gene interactions exist. For example MDR [10] is a method for exhaustive search of high-level multi-locus interactions, although it is extremely computationally intensive, and genome-wide searches for epistasis are prohibitive. More recently a promising Bayesian method (BEAM) has been suggested as a powerful alternative for detecting epistatic interactions, although it is not exhaustive and still requires further improvements to effectively handle the large SNP datasets commonly used in genome-wide studies [11]. Other methods, like PLINK [9] or others based on logistic regression [5], can carry out genome-wide epistasis tests relatively quick, but are currently limited to two- or three-locus models, and only perform general tests of epistasis. HFCC combines a relatively fast computing algorithm for genome-wide epistasis detection, with the flexibility to test a variety of different epistatic models in multi-locus combinations. Our analysis of a simulated dataset reveals that HFCC has good power, at least as good as or better than MDR [20], to detect epistatic interactions, as long as they are relatively strong and common. In the most extreme simulation, with 5% genotyping error, 5% missing data, 50% phenocopies, and 50% genetic heterogeneity, HFCC still had 71% power to detect some types of epistasis, although the power for other types of epistasis was smaller (34–51%). We will need to carry out more extensive simulations to evaluate the power under different conditions and genetic models.For this illustrative application of the software we have also analyzed an open-access dataset of Parkinson's disease patients and unaffected controls. We would like to emphasize here the importance of these public datasets of real data to improve the quality of applied research, and also to foster the development of new methodology.One of the pecularities of HFCC is the possibility of dividing the case-control sample into replication groups, to detect only those effects that are consistent across samples. The number of replication case-control groups to be used depends on the available dataset (sample size and number of genetic markers). It is an important analysis parameter because the overall significance level is a function of the selected critical value for the test statistic and the number of replication groups. For the current analysis, the sample of cases was divided into three replication groups, and so were the controls. This strategy focuses on the detection of large and consistent effects, which are hopefully detectable with the available datasets. It is reasonable to expect that the joint effect of a combination of genes is larger and more penetrant than each of the single-locus effects, and therefore under some circumstances (i.e., not extremely heterogeneous or rare effect), these multi-locus effects can be identified. The detection of small, rare or heterogeneous effects may need larger samples and more complex models.HFCC allows for a variety of different genetic models and tests. Different models may be necessary to detect different types of effects, such as recessive, dominant or heterozygote effects. The best analysis strategy may depend on prior knowledge or hypothesis about the trait. An optimal strategy would apply a selected subset of models which would maximize the chances of detecting a hypothesized effect. For example, for this Parkinson's disease study, we have employed a subset of nine epistatic models which typically detects recessive effects.Another characteristic of HFCC analysis is the successive application of noise-signal filters. Control groups can be compared against each other to remove background noise associations. Direction of effect can be taken into account, so that only those results consistent in strength and direction across replication samples are selected. A final filter is able to remove those multi-locus results which are primarily due to quality-control failing markers (ie, in Hardy-Weinberg disequilibrium, low allele frequency or low call rate) or to large single-locus effects. The remaining multi-locus combinations can be categorized into epistatic, conditional and simultaneous effects, and interaction tests can be used to detect possible epistatic interactions over and above the marginal effects. The selected markers can then be included in a validation analysis in an independent sample. As an illustration, Table 4 displays 30 two-locus combinations suggestive of displaying epistatic interactions influencing the development of Parkinson's disease. It is important to note that due to the small sample size used in this experiment, these results may not be reliable, and need re-analysis or confirmation in larger datasets. The number of combinations selected for a validation analysis depends on many factors, and tools are included to help perform this selection.The Parkinson's disease study reported here can illustrate several issues regarding the search for epistatic effects in large datasets. One of the most difficult tasks in large dataset analysis is selecting the most promising candidate results. The huge number of statistical tests performed requires a severe statistical cut-off, or a protocol of data filtering, to be able to select only the most promising results. For example, the two-locus analysis of a genome-wide association SNP dataset presented here reveals several hundred thousands two-locus marker combinations at a liberal significance level (i.e., p value < 10-6). Using a stringer significance level, such as a Bonferroni correction, may be overly conservative, sometimes potentially missing real effects. HFCC filters and post-hoc analyses help selecting the most promising two-locus interactions from a large set of preliminary findings. For example, all two-marker combinations in Table 4 are consistently associated with Parkison's disease in three replication groups (overall two-locus X2 (1 df) in the range 21–30), and they all also deviate from a multiplicative model (case-only X2 (1 df) in the range 7–33) suggesting an epistatic interaction.The resampling validation analysis raises an important issue regarding the difficulty in replicating a result across different validation samples, a finding that may reflect the general lack of power to detect these types of effects, especially in the presence of heterogeneity. With the available sample size for this study we have approximately 80% power to detect large common effects (Odds ratio > 3 in a genotype prevalence > 25%) at a significance level of 0.01 per replication group. However, this small dataset is underpowered to find more moderate, and perhaps more realistic, effect sizes. The general lack of consistency suggested by our own sensitivity analysis may be a consequence of the small sample size analyzed. Fung et al. (2006) claimed that there is no common genetic variant that exerts a large genetic risk for late-onset Parkinson's disease in white North Americans. Multi-locus analysis may, however, reveal the existence of large complex (multi-locus) genetic effects.Analysis GuidelinesHFCC provides a tool for the genome-wide study of epistasis. Its use may depend heavily on the researcher's goals and the data available. For this reason, it is hard to provide general guidelines on the optimal parameters for analysis, but HFCC's flexibility to accommodate to the specific needs of each study is a great asset.A key parameter is the number of replication groups. When the available sample size is fixed, dividing the sample into more replication groups decreases the power of the analysis, but also increases the confidence in the remaining results. For example, the two-locus analysis of the full Parkinson's dataset in one case-control group with a Type I error set at 10-6 yields a total of 784,506 preliminary positive results. The analysis of the same data splitted in three replication groups, each with alpha = 10-2, yields only 418,535 results (53% of the single-group results).There is not an optimal number of replication groups. Researchers need to select this parameter as well as the significance level cut-off to fit their dataset and study design. For example, a study of three related diseases or phenotypes suggests the use of three replication groups. In the case of a single disease, the number of replication groups, the sample size in each group, and the statistical cut-off should be chosen depending on the nature of the study. A strict statistical correction may be necessary if the results are to be conclusive, while a more relaxed criterion may be used in a two-stage study where the goal of the preliminary analysis is to select a subset of markers for subsequent validation.HFCC's flexibility is also possible in the application of data filters. The results displayed in Table 2 suggest that the control filter and the direction filter can eliminate most of the same noise results. The application of either or both of these filters can therefore depend on the study design. If a study has many control subjects and a limited number of cases, it can benefit from using the control filter. If a study has many affected individuals, then several replication groups and the direction filter can be used.There are also 255 possible two-marker genetic models that can be tested. Many of them are correlated, so it is probably not necessary to test them all. We suggest using a small subsample of models that cover the researcher's hypothesis. For example, for the Parkinson's analysis in this paper we tested nine simple genetic models. To identify subsamples of models that may optimize the chances of discovering epistatic effects of different nature would need a thorough simulation analysis that is beyond the scope of the current paper. Another analysis option is to use more general tests of epistasis, which are also implemented in the software.Statistical IssuesThe two-locus analysis of the full Parkinson's dataset as presented here comprises a total of 708 × 109 statistical tests. The two-locus analysis of these data in one case-control group with a Type I error set at 10-6 yields a total of 784,506 preliminary positive results, about 10% more of those expected by chance. This false positive inflation is probably due to the tracking markers (mainly QC-fail markers) as well as to the correlated nature of some of the statistical tests, and can be controlled by the use of replication groups, or by applying more stringent cut-offs if necessary.For the simulated dataset, analyzed with only one case-control group a significance level of 10-2 (chi-square 6.64), the Type I error was actually lower than expected (approximately around 0.006 on average). These results demonstrate that HFCC analysis is not only powerful but can also be conservative, preserving against Types I and II Errors. More thorough simulations to assess the impact of sample size, allele frequency, number of replication groups, and noise filter applications are needed in the future to understand these issues in detail.Applying strict multiple testing correction (Bonferroni) to the Parkison's disease analysis, we do not find any significant two-locus interactions. The reason for this may be the small sample size available in the Parkinson's dataset. But we can still select the most promising two-locus results for subsequent validation. The key issue is whether we are concerned with achieving an absolute level of statistical significance, which may not be properly defined in this setting given the complexity of the analysis suggested (multiple testing of correlated hypotheses, independent replication of results, data filtering steps), or selecting the most promising markers or marker combinations that pass a more or less stringent statistical criterion. In either case, the usefulness of HFCC for selecting marker combinations for later validation is doubtless.Computational IssuesMulti-locus analysis is computationally intensive and is therefore limited by computing capabilities. HFCC is a relatively fast algorithm considering the huge number of computations it performs. The dataset analyzed in this study consists of 396,591 genetic markers, which results in 78.6 × 109 two-locus marker combinations. Nine genetic models were tested in the current study, resulting in a total of 708 × 109 statistical tests. Moreover, these tests may be carried out in as many as three case-control replication groups, and also in as many as three control-control noise-filter groups, so the computing task is staggering. HFCC is programmed to take advantage of computer resources by dividing the computing task into processes which can migrate to the available CPUs in a computer server or cluster. For the current analysis, we employed a computer cluster consisting of twelve 3.2 GHz CPUs, which was able to carry out the full genome-wide analysis in approximately 5 days.Another computational complication for multi-locus analysis is related to RAM memory. The data matrices need to be loaded onto memory to speed computations, and therefore a limitation exists for the analysis of very large datasets (millions of genetic markers or several thousands of subjects) where RAM memory is rapidly exhausted. This limitation, however, can be solved with parallel processing techniques (MPI like), such as dividing the data matrices into smaller subsets which are distributed around the computer network.HFCC can perform more complex multi-locus analysis (3-locus, 4-locus, etc.), but the number of computations grows exponentially with the number of interacting markers, and the analysis becomes dependent on computing resources and time limitations. Depending on these resources, genome-wide three- or four-locus analysis may require a two-stage strategy, where some markers are selected first by single-locus analysis, and then employed to guide the multi-locus testing [19]. Our exhaustive two-locus genome-wide analysis of a Parkinson's disease dataset reveals that pure epistatic effects, as defined here, are rare (0.01% of the preliminary results). Therefore, a two-stage strategy for multi-locus analysis may be a more economical analysis with minimal information loss. This statement assumes that we had power to detect these epistatic effects, that more complex interactions behave as two-locus ones, and that Parkinson's disease is representative of other diseases. Our results suggest the use of a conditional two-stage strategy, where a liberal single-locus threshold is first used to select loci with marginal effects, and then these markers are used against the full panel for multi-locus analysis. This conclusion is similar to some previous suggestions [5,19] but not all [21], confirming that a liberal single-locus cut-off (i.e., p < .05) greatly reduces the computational task while minimizing the probability of discarding potential epistatic loci.It is also important to note that linkage disequilibrium (LD) is unaccounted for in our analyses. LD reflects an association among markers and therefore can affect the results of some tests. For example, it can produce a significant case-only chi-squared test. Nonetheless, HFCC's algorithm and analysis filters seem to prevent this bias. In the case where one marker is associated with several markers in LD, these results are detected in the last stage of marker selection.ConclusionIn summary, we propose that genome-wide multi-locus analysis is performed on available datasets of common diseases, because they can exploit the large genetic datasets and computing resources becoming available, to open a new phase of genetic analysis. The analysis of Parkinson's disease reported here represents the first exhaustive genome-wide epistasis search on a real dataset, effectively handling hundred of thousands of genetic markers, and demonstrating its feasibility. Due to the small sample size, however, these results are only illustrative and require re-analysis or confirmation in larger datasets. These multi-locus analyses would not substitute conventional single-locus analysis but add a new layer of genome-wide association studies, allowing the identification of new candidate markers for further validation. HFCC is a new genome-wide multi-locus software, which allows the user a high degree of control over analysis parameters, so that data analysis can be tailored to the specific needs of each project. HFCC can have a great impact on the discovery of the genetic causes of common diseases, especially to identify those multi-locus effects that may not be detectable using the available single-locus methods. The discovery of new genes affecting a disease may be useful as predictive tools or to find new therapeutic targets. HFCC has multiple applications, not only in the study of disease phenotypes, but also of other qualitative traits, and can be used in clinical trials or pharmacogenetics studies.Availability and requirementsHFCC is written in C and freely available for linux platforms from this Website: List of abbreviationsCPU: central processing unit; GE: genotyping error; GH: genotyping heterogeneity; HFCC: hypothesis free clinical cloning; HWE: Hardy-Weinberg equilibrium; LD: linkage disequilibrium; MS: missing data; PC: phenocopy; SNP: single nucleotide polymorphism; XOR: logical exclusive \"or\" statement.Authors' contributionsJG, AG–P, and AR conceived and designed the experiments. JG, AG–P and AR analyzed the data. JG and AR wrote the paper. AR conceived the analysis tool. All authors helped develop the analysis tool. FB and AQ wrote the analysis tool. All authors read and approved the final manuscript.Competing InterestsAs a declaration of competing financial interest, authors in the paper are employees and/or shareholders in Neocodex. Neocodex owns a patent on  the HFCC algorithm described in this paper (WO 2008010195 20080124).\n\nREFERENCES:\nNo References"
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batch_11/PMC2533063.json

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+{
+    "id": "PMC2533063",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2533063\nAUTHORS: Kasturi Haldar, Grant McFadden\n\nABSTRACT:\nNo Abstract\n\nBODY:\nAs PLoS Pathogens turns three, we are pleased to say that the publication is flourishing. A journal's overall health can be measured in numerous ways: high quality and breadth of the research; high citation counts of individual papers; solid submission numbers; rising impact factor; wide influence and robust discussion within the community; strong editorial leadership; rapid publishing speed; accessibility of research; and, in our case especially, an unwavering commitment to creativity and new facets of open-access (OA) publishing. In all of these areas, the journal has grown and gained strength in the past three years through the collaborative effort of the community, editorial board, and PLoS staff to produce a publication that is both innovative and integrative.The crux of PLoS Pathogens' strength lies in its vision and commitment to publish “outstanding original articles that significantly advance the understanding of pathogens and how they interact with their host organisms” and to make those articles immediately and widely available. By publishing a broad range of topics, including viruses, bacteria, prions, yeast and fungi, and parasites, readers can cross-reference methodologies and findings across various disciplines that traditionally have been segregated into specialist journals. This provides not only a breadth of information but also unique interdisciplinary consultations among outstanding editors whose expertise crosses both pathogens research efforts and the various scientific communities.As the journal completes its first three-year editorial term, we especially thank the editorial board for their willingness to make a sustained commitment to PLoS Pathogens and the quality of work we strive to publish. We also thank the pathogens community for their groundswell of support, as evidenced in a trend of record manuscript submissions each month. This support in turn drives the need to continue to build a diverse editorial board that is integrated with its members' imperative to publish research of the highest caliber with significance across pathogens.\nPLoS Pathogens is committed to OA publishing, making all of our content freely available to read, download, and reuse. The business model for and opportunities available through OA publishing are still evolving, but as funding agencies and academic institutions around the world advance laws, mandate actions, and launch key initiatives to support full access to published scientific articles, PLoS Pathogens' reach will only continue to expand.This year PLoS Pathogens also provides a unique outlet among pathogens research journals with its transition to a new Web publishing platform, known as Topaz. Accepted manuscripts are now published more rapidly, and the online functionality enables members of the community to interact with the science and each other through erudite discourse on the published articles. This discussion is overseen by our new team of Community Editors, who encourage and review responses to the published articles—fostering a new method of increasing the journal's utility, accessibility, value, and ultimate impact on the field.Thus, in three years, PLoS Pathogens has established itself as a leading journal that is poised to grow further. We are proud of what has been achieved so far, in such a short period of time, and we welcome the opportunity to publish your best research on pathogens and their host interactions.\n\nREFERENCES:\nNo References"
+}

batch_11/PMC2533097.json

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+{
+    "id": "PMC2533097",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2533097\nAUTHORS: Yau-Hua Yu, Hsu-Ko Kuo, Kuo-Wei Chang\n\nABSTRACT:\nBackgroundNumerous studies were performed to illuminate mechanisms of tumorigenesis and metastases from gene expression profiles of Head and Neck Squamous Cell Carcinoma (HNSCC). The objective of this review is to conduct a network-based meta-analysis to identify the underlying biological signatures of the HNSCC transcriptome.Methods and FindingsWe included 63 HNSCC transcriptomic studies into three specific categories of comparisons: Pre, premalignant lesions v.s. normal; TvN, primary tumors v.s. normal; and Meta, metastatic or invasive v.s. primary tumors. Reported genes extracted from the literature were systematically analyzed. Participation of differential gene activities across three progressive stages deciphered the evolving nature of HNSCC. In total, 1442 genes were verified, i.e. reported at least twice, with ECM1, EMP1, CXCL10 and POSTN shown to be highly reported across all three stages. Knowledge-based networks of the HNSCC transcriptome were constructed, demonstrating integrin signaling and antigen presentation pathways as highly enriched. Notably, functional estimates derived from topological characteristics of integrin signaling networks identified such important genes as ITGA3 and ITGA5, which were supported by findings of invasiveness in vitro\n[1]. Moreover, we computed genome-wide probabilities of reporting differential gene activities for the Pre, TvN, and Meta stages, respectively. Results highlighted chromosomal regions of 6p21, 19p13 and 19q13, where genomic alterations were shown to be correlated with the nodal status of HNSCC [2].ConclusionsBy means of a systems-biology approach via network-based meta-analyses, we provided a deeper insight into the evolving nature of the HNSCC transcriptome. Enriched canonical signaling pathways, hot-spots of transcriptional profiles across the genome, as well as topologically significant genes derived from network analyses were highlighted for each of the three progressive stages, Pre, TvN, and Meta, respectively.\n\nBODY:\nIntroductionHead and Neck Squamous Cell Carcinoma (HNSCC), ranked the 6th common cancer worldwide, has long been recognized with a heterogeneous clinical presentation and a poor prognosis in advanced stages [3]. Historically, decisions to pursue aggressive treatments such as chemoradiotherapy (CRT) or neck dissection, to a very large extent, depended on clinical staging. Many patients suffered from treatment failure due to local recurrence, distant metastases, or the development of second primary HNSCC, even with the utilization of multi-modality treatment options [4]. Nevertheless, understanding mechanisms of tumorigenesis and metastatic progression remains as the most urgent call for future remedy of HNSCC.Recent advance in using microarray technology to investigate HNSCC cancer biology has attracted significant research interest. Choi and colleagues [5], who took the lead in systematically summarizing the HNSCC transcriptome, discovered such significant biological pathways as cell cycle regulation, inflammatory response, mevalonate pathway, and down-regulation of genes encoding cytoplasmic ribosomal proteins. These reconfirmed or newly discovered pathways shed light on the pathophysiology of HNSCC.Graphical presentations of biological networks become a common platform to demonstrate gene–gene interplays and to model human diseases at systems-level [6]. Integrative meta-methods, which utilize genomic, proteomic and phenotypic information from various sources, have been shown reliable in generating novel experimental hypotheses [7], [8]. However, an integrative approach of network analysis and systems biology has not been applied to HNSCC.We recently developed a knowledge-based network approach to conduct genomic meta-analysis of HNSCC [9]. This article, aiming to depict the evolving transcriptome of HNSCC via comparisons of gene expression profiles, sought to dissect the progressive states of HNSCC by examining the following three comparisons: Pre, premalignant lesions v.s. normal; TvN, primary tumors v.s. normal; and Meta, disseminated (regional lymph node involvement, distant metastases, or local recurrence) v.s. primary localized tumors. Results in the original articles were demonstrated to identify patterns of etiologic or metastatic processes of HNSCC in terms of Pre, TvN, or Meta. Here, we synthesized results of differential gene expression profiles into knowledge-based interacting networks. It was designated that the evolving nature of HNSCC could be read out from the topological characteristics, the prismatic visualization of three staged interacting networks, and the probabilities of reporting differential gene activities across the genome.MethodsSearch StrategyWe conducted this meta-analysis in accordance with the standard protocol recommended by the Quality of Reporting of Meta-analyses group [10]. A systematic search in the PubMed database (Jan 1994 to Apr 2008) was performed using a complex query, consisted of keywords “head and neck neoplasm”, “gene expression profiling”, “oligonucleotide array sequence analysis”, “microarray” and “carcinoma, squamous cell”. Details of the complex query were provided in Text S1. A total of 410 potentially relevant articles were identified. First, we excluded studies examining lesions located in thyroid glands (n = 82), salivary glands (n = 16), nasopharynx (n = 23), eyes or elsewhere (n = 5). Studies were further excluded if the primary data reported was not differential gene expression profiling (n = 118), or if the samples used in the experiment were not human tumor tissue (n = 96). Of the remaining 70 studies, six were examining HPV-related or smoking-related transcriptional profiles and one was unavailable. Sixty-three studies of transcriptional profiles of HNSCC were included in the meta-analysis and classified into three specific comparisons: Pre, premalignant lesions v.s. normal (n = 5); TvN, primary tumors v.s. normal (n = 41); and Meta, disseminated (regional lymph node involvement, distant metastases, or local recurrence) v.s. primary localized tumors (n = 26). Figure 1 demonstrated the QUOROM flow diagram of screening microarray-based HNSCC transcriptional profiles.10.1371/journal.pone.0003215.g001Figure 1QUOROM flow diagram of the systematic reviews and meta-analysis.The diagram summarized the search strategy. In order to be included, studies had to examine the HNSCC tumor samples by means of microarray-based gene expression profiling.Data extraction, processing and parsingFor each study, reported genes were extracted from tables, text or supplements. We converted various kinds of gene or sequence identifiers, such as UniGene cluster IDs, Genbank accession numbers, gene symbols, or Affymetrix probe sets, into the universal Entrez GeneIDs. GeneIDs conversion was done by the web-based program of DAVID2007–2008 (Database for Annotation Visualization and Integrated Discovery, NIH) [11]. Then, for each article, we compiled information of the PubMed ID, the original identifiers, the converted Entrez GeneIDs, and the values or directions of fold changes into a standard format (Text S1). Program scripts were developed for the use of ActivePerl and R statistical software (version 2.6.1) in text processing, parsing networks (svg files), analyzing and comparing the gene lists, linking gene-specific information and chromosomal coordinates, and transforming gene-gene interactions into matrices for networks topological analyses. Cytoscape software (version 2.5.1) was used for the illustration of networks [12].Quantitative data synthesis: bounded fold changesIn order to synthesize the original information of fold changes, a novel method was developed to overcome differences in multiple microarray platforms. This method based on the assumption that different transcriptional measurements of the same target gene, i.e. different sequence or gene identifiers converted into the same Entrez GeneID, were indeed representing the same functional entity. For each study, if fold changes, θ, were reported, the standardized bounded fold changes would be |θ′| = θ/(maximum of θ) with regard to up- and down-regulated genes separately; alternatively, if only directions of up- or down-regulation were reported, the bounded fold changes would be translated into 1 for up and −1 for down, respectively. For each of the Pre, TvN or Meta stages, consensus of gene expression was computed as the averaged mean of the bounded fold changes if studies reported values of fold changes; else, the median was computed instead if at least one study reported only directions of gene regulation. Genes without such information were coded with 0 as the bounded fold changes in the analysis.Validity assessmentOwing to the underlying heterogeneity among included studies, such as differences in the tumor of origins (oral, pharynx, or larynx), multiple microarray platforms, different analytical approaches taken, and diverse endpoints, we sought to examine the validity of three staged classification of the HNSCC transcriptome. First, we systematically computed the frequency of reporting the same Entrez GeneID within each stage of comparison. If a gene was reported more than once, this gene was regarded as verified. Subsequently, the validity of TvN and Meta was tested through comparisons of ratios of internal consistency, defining as the percentage of verified genes for each study within the groups of interest, i.e. studies of lesions in different subsites, studies using different microarray platforms, studies investigating different endpoints, or the proposed classification. Classifying studies in TvN or Meta would be considered reasonable if the ratios of consistency were higher than those within the subgroups of interest. For instance, of the 121 differentially expressed genes by Ibrahim and colleagues, 74 genes (61%) were verified when classified in TvN; 38 genes (32%) verified among the 23 studies using cDNA microarrays in TvN; as well as 32 genes (27%) verified among the 19 studies examining tumors located exclusively in the oral cavity.Knowledge-based network analyses and identification of enriched canonical pathwaysThe bounded fold changes of the full gene lists and the verified gene lists of three comparisons, Pre, TvN, and Meta, were imported into the Ingenuity Pathways Analysis (IPA) Software (Ingenuity Systems, Redwood City, CA, USA) to obtain six sets of networks for further analyses. We used IPA to identify the top 10 enriched canonical pathways for each stage of comparison (Fig. 2). The enriched canonical pathways were ranked by the p-values of the Fisher's exact test, which indicated the probabilities of the input genes to be associated with genes in the canonical pathways expected by chance.10.1371/journal.pone.0003215.g002Figure 2Ranking of the enriched canonical pathways.Eight canonical pathways were found in consensus in IPA with the full gene lists (all) or the verified gene lists (fq2) as inputs. Embedded table showed rankings of the canonical pathways in different analyses. Antigen presentation, calcium signaling, and integrin signaling pathways were highlighted as highly enriched across three progressive stages. For each stage of the HNSCC transcriptome, figure panels demonstrated the enriched canonical pathways ranked by the −log (p-values) (right y-axis), that is, the orange line with square data points. Colored bars were indicating the percentage (left y-axis) of the up- or down-regulated genes within each canonical pathway. The numbers on top of the colored bars were the number of total genes in the canonical pathways.Networks generated from the IPA consisted of identified focus genes from the user input and other correlated molecules or genes from the knowledge base [13]. Networks were scored and ranked according to the probabilities of having more focus genes than expected by chance. To gain a comprehensive perspective of the HNSCC transcriptome, three sets of networks generated from the IPA analyses with the full gene lists as inputs were parsed and merged into three staged interacting networks, representing the Pre, TvN, and Meta progressive states of HNSCC. Only networks of scoring higher than 10 (log p-value) were included in the analysis.Network topological analysesBased on findings in model organisms, indicating that network structure such as dynamic modularity [14] and topological cartography [15] determined the key aspects of regulation and functionality, we developed a new framework of network topological analyses to estimate the implied pathological effect for each gene in the HNSCC transcriptome. The idea was to evaluate the functional significance of a gene based on the concept of connectivity. An inter-modular hub-designated as a cut node in the graph - would cause the original component breaking down into different blocks upon removal of itself, leading to the blockade of signaling crosstalk. If an inter-modular hub i was disrupted, i.e. removed in the interacting network, we estimated the potential for pathophysiological perturbation by the informational score (fi), computed by the number of blocked out paths divided by the number of breaking down components. The informational score (fi) was calculated as equation (1), where m denoted the number of broken-down components after removing a node i, Ncp denoted the total number of nodes in this component where node i was located, S was the size of each broken-down component, and m.lg was the iterating counter part for each S to calculate the blocked-out paths.(1)Rest of the genes were grouped into the periphery genes or the intra-modular hubs, whose interactions were equal or greater than two and whose removal would NOT cause the breakdown of networks. We did not consider the pathophysiological effects of these two groups of genes based on the topological properties.Sub-networks of enriched canonical pathways were extracted from each of the three progressive stages of the HNSCC transcriptome for independent topological analyses (Fig. 3a–i). Prominent connecting hubs were identified by the topological characteristics, changes in the transcriptional profiles, or the participation among different stages of the HNSCC transcriptome.10.1371/journal.pone.0003215.g003Figure 3Enriched antigen presentation and integrin signaling pathways in each stage of comparison of the HNSCC transcriptome.Genes were represented by nodes and functional associations by edges. Node coloring was scaled to the bounded fold changes–red: up-regulated; green: down-regulated. Node size was proportional to the number of papers reporting this gene in the HNSCC transriptome. Edges were colored according to the stage of the HNSCC transcriptome–red: Meta; blue: TvN; and green: Pre. a–c. Merged networks of the antigen presentation pathways with nodes colored according to the bounded fold changes in Pre, TvN, and Meta, respectively. d–f. Merged networks of the integrin signaling networks with nodes colored according to the bounded fold changes in Pre, TvN, and Meta, respectively. g–i. Sub-networks of the integrin signaling pathway in the Pre, TvN and Meta stages of the HNSCC transcriptome. (For details, please see Figure S1, S2, S3, S4 and S5).Probabilities of reporting differentially expressed genes within each cytoband along the chromosomal coordinatesAcross the genome, we computed the accumulative probabilities (PB) of reporting genes within the chromosomal segments for each stage of comparisons. For instance, in the TvN comparison consisted of 41 papers, the cumulative probability of a cytoband with k genes reported would be , where fqg was the number of articles reporting gene g in TvN. In order to compare across three progressive stages, we standardized PB of the Pre, TvN, and Meta to be within 0∼1 by dividing the maximum probability of each stage. We plotted PBs of three progressive stages along the chromosomal coordinates to identify hot-spot regions. For the identified hot-spots, differential gene activities (bounded fold changes) were illustrated on the resolution of a single gene for each stage of comparison.ResultsConsensus among genes reported in each stage of comparisonThere were 1822, 4311, and 2293 genes, respectively, reported from 5, 41, and 26 papers in the Pre, TvN, and Meta comparisons. Eighty-two genes out of 1822, 1260 out of 4311, and 321 out of 2293 were found reported at least twice in the Pre, TvN, and Meta comparisons. With regard to the direction of fold changes of the verified genes, we found the least contradiction in TvN (217/1260 = 17.2%), less in Meta (117/321 = 36.5%), and the most, in Pre (42/82 = 51.2%). There were few genes overlapped between Pre and Meta, whereas many overlapped between any other pairs of comparisons. In Table 1, reported genes were listed according to the number of reporting studies for each stage of comparison. Notably, MMP1, reported 13 times in TvN with a bounded fold change of 1, was found consistently highly up-regulated, albeit that tumors were harvested from different anatomical subsites; whereas in the Meta comparison, we found MMP1 with three studies reporting induced [16], [17], [18] and one repressed [19]. We speculated that the contradictory regulation of MMP1 in Meta might stem from differences in prognostic endpoints investigated rather than the lesion sites.10.1371/journal.pone.0003215.t001Table 1Highly reported genes with bounded fold changes in each stage of comparison.PreTvNMetagenefqchrfoldgenefqchrfoldgenefqchrfold\nNR2F2\n315q260.14\nKRT4\n1812q12−0.73\nTNC\n69q330.03\nEMP1\n312p12.30.14\nKRT5\n1612q12−0.72\nPI3\n520q12−1\nTAP1\n36p21.30.16\nPLAU\n1510q240.48\nSFRP4\n47p14.10.19\nCOL6A3\n32q370.14\nFN1\n142q340.45\nPLEC1\n48q240.51\nGPRC5A\n312p13−0.46\nMAL\n142cen-q13−0.96\nMMP1\n411q22.30.6\nKRT1\n212q12−0.35\nMMP1\n1311q22.31\nTRIM22\n411p15−0.46\nDPYSL3\n25q32−0.42\nCOL1A2\n137q22.10.4\nSERPINB2\n418q21.3−0.32\nCRIP1\n214q32.3−0.41\nSPARC\n135q31.30.43\nFN1\n42q340.44\nCXCL10\n24q210.67\nPOSTN\n1213q13.30.61\nPOSTN\n413q13.30.71\nIGJ\n24q210.82\nIFI6\n121p350.33\nDSG3\n418q12.1−0.44\nRBP1\n23q230.59\nTGM3\n1220q11.2−0.86\nFGFBP1\n44p16−0.19\nIFI44\n21p31.10.58\nSPP1\n114q21-q250.79\nEGFR\n47p12−0.29\nCA2\n28q220.56\nITGA6\n112q31.10.26\nTGM3\n420q11.2−0.89\nADH7\n24q23−0.16\nKRT13\n1117q12-q21−0.53\nPLAU\n410q241\nCOL4A1\n213q340.33\nEMP1\n1112p12.3−0.48\nITGB4\n417q25−0.32\nCOL6A1\n221q22.30.22\nECM1\n111q21−0.57\nCHPT1\n312q−0.11\nLOX\n25q23.20.18\nTNC\n109q330.43\nCDH3\n316q22.10.32\nFKBP1A\n220p130.18\nMMP10\n911q22.30.75\nKRT16\n317q12−1\nSEC23A\n214q21.1−0.44\nMMP3\n911q22.30.59\nPHC2\n31p34.30.23\nCTNND1\n211q11−0.03\nMMP12\n911q22.30.55\nRAC2\n322q13.10\nCLINT1\n25q23.1−0.03\nLAMC2\n91q25-q310.55\nGREM1\n315q131\nTHY1\n211q22.30.26\nIL8\n94q13-q210.73\nGPC5\n313q32−1\nCOL4A2\n213q340.33\nKRT17\n917q120.39\nKRT14\n317q121\nAK2\n21p340.01\nCOL5A2\n92q14-q320.43\nMMP2\n316q131\nKRT10\n217q210.13\nCOL4A1\n913q340.37\nLGALS1\n322q13.11\nAbbreviations: fq, the number of papers reporting a gene; chr, the chromosomal coordinate; fold, the consensus of the bounded fold changes.Validity of the TvN and Meta progressive stages of the HNSCC transcriptomeWe found a higher internal consistency, i.e. having a higher ratio of the verified genes, among studies in TvN (mean ratio = 71%), secondly in Meta (31%), and lastly in Pre (26%) (Table 2). In TvN, we further investigated ratios of consistency within subgroups of studies examining tumors from distinct anatomical subsites (p, pharynx; L, larynx; and o, oral cavity) or tumors from unspecified locations (mix). Nineteen papers procured specimens located exclusively within the oral cavity. Unexpectedly, we found a significant decrease of the internal consistency within this subgroup (mean ratio = 37%). Gene lists reported from distinct anatomical subsites were examined in details (Text S1). The majority of genes reported from tumors of distinct oral cavity (63%), pharynx or larynx (74%) overlapped with those from unspecified locations. There were 94 genes reported in common from distinct and mix groups; 58 genes specific to pharynx or larynx; and 376 genes specific to oral cavity. Particularly, 98 genes, accounting for 9.6% from oral cavity as well as 41% from pharynx or larynx, overlapped and suggested that some similarities existed among the distinct groups. We also noted decreases of internal consistencies within subgroups of studies using similar microarray platforms. In general, studies using Affymetrix Genechips demonstrated higher internal consistencies. Three studies using Affymetrix HG-U95A and 4 studies using Affymetrix HG-U133 held with more overlap in the findings; however, another four studies using Affymetrix HG-U95Av.2 did not. Taken cDNA microarrays as a single category, four studies remained at the same level of consistency while the rest showed considerable decreases (data not shown). It was of importance to be reminded that the size of the reported gene lists would certainly affect the ratio of consistency. Details of the numbers and anatomical subsites of procured tumor samples, microarray platforms used, analytical methods conducted, original identifiers reported, information regarding fold changes, and the availability of datasets were provided in Table S1.10.1371/journal.pone.0003215.t002Table 2Ratios of internal consistency of selected studies in each stage of comparison.Articles in TvNratioratiositeArticles in PreratioratiositeYe et al289/3160.91oKondoh et al9/270.33oSuhr et al92/175l0.53oBanerjee et al69/13480.05oBraakhuis44/590.75mixOdani et al11/180.61oZiober et al68/760.89mixCarinci et al21/1590.13oKainumai et al8/100.8mixHa et al59/3570.17mixGottschlich et al12/220.55pLTomioka et al32/460.7o\nArticles in Meta\n\nratio\n\nratio\n\nsite\n\ncategory\nDysvik et al29/500.58mixMendez et al28/1800.16opJarvinen et al16/400.4LPramana et al9/400.23mixrRoesch Ely et al5/51mixCarinci et al9/380.24LpBelbin et al107/2080.51oChung et al17/440.39mixsSchlingemann et al46/630.73pVachani et al53/920.58mixdKornberg et al91/1130.81opZhou et al16/480.33opCarinci et al7/270.26oNguyen et al16/680.24opLaytragoon-Lewin et al8/120.67mixKato et al3/190.16opChin et al26/440.59opRoepman et al167/7420.23mixpShimada et al2/90.22oCarinci et al20/1250.16odIrie et al6/100.6oBelbin et al53/2080.25opCromer et al119/1250.95pO'Donnell et al6/290.21opSchmalbach et al48/570.84oRoepman et al71/960.74mixpGinos et al902/21260.42mixIrie et al13/200.65opMarcus et al45/600.75opChung et al70/1800.39mixp,sToruner et al43/540.8oSchmalbach et al28/570.49opKuriakose et al40/401mixWarner et al0/150opTsai et al48/620.77oNagata et al14/200.7opWhipple et al44/470.94opBraakhuis et al10/410.24mixdHa et al875/20410.43oLGiri et al8/390.21mixdBanerjee et al5/51oTalbot et al14/650.22mixdNagata et al31/350.88oCromer et al9/360.25pdSok et al195/2310.84mixGinos et al22/590.37mixrGonzalez et al5/70.71oGanly et al4/160.25mixsLeethanakul et al26/370.7oWinter et al30/1540.19mixsKuo et al2/90.22oBelbin et al29/2600.11mixsIbrahim et al74/1210.61oHwang et al38/43l0.88oEl-Naggar et al8/110.73opMendez et al244/3050.8opSquire et al11/130.85oAlevizos et al41/420.98oLeethanakul et al33/570.58mixVillaret et al10/130.77mix\nAbbreviations: o, oral; p, pharynx; L, larynx; op, oral and pharynx; oL, oral and larynx ; pL, pharynx and larynx; mix, multiple tumor of origins; p, positive lymph node; r, recurrence; d, distant metastasis; s, survival.\nNote: detailed references were provided in Table S1.With regard to differences of prognostic outcomes investigated in Meta, we classified studies into 4 subgroups (pN, positive lymph nodes; recur, recurrence; dM, distant metastasis; and surv, survival). Similar to the results in TvN, ratios of internal consistencies within each subgroup dropped significantly, especially in the dM and surv group (mean ratio = 23%, 5%, 5% for pN, dM, and surv subgroups, respectively). Overall, we believed that classification of three progressive stages, Pre, TvN and Meta, of the HNSCC transcriptome was sufficient for the purpose of this meta-analysis.The global HNSCC transcriptome in Pre, TvN, and MetaFor reasons of the inherent noise in the HNSCC transcriptome, we developed a strategy to capture the essential themes by focusing on the enriched signaling pathways as well as the topological properties of the interacting networks. In the verified gene lists, findings were more stably shown in HNSCC but the scope might be too limited. In contrast, using the full gene lists might facilitate findings of significant modules, which might be obscure when data were scarce. Therefore, the bounded fold changes of the full gene lists and the verified gene lists of Pre, TvN, and Meta were first imported into IPA to obtain six sets of networks. We compared these two levels of analyses to come up a consensus of the enriched signaling pathways. The interacting networks of the HNSCC transcriptome were ultimately built on those generated by the full gene lists. Networks of scoring higher than 10 (log p-value) were parsed and merged into three staged interacting networks, representing the Pre, TvN, and Meta progressive states of HNSCC.\nTable 3 described statistics of three staged interacting networks. A total of 65, 100 and 64 networks were merged into the Pre, TvN and Meta interacting networks, respectively. The density (average degree) of the networks was similar for each stage, and all of them contained a major component (a connected subset) of the size around 2000 genes. We did network topological analyses and identified significant inter-modular hubs with the highest informational scores. Likewise, intra-modular hubs of the highest connectivity for each stage of comparison were also listed in Table 3.10.1371/journal.pone.0003215.t003Table 3Network Statistics.StagePreTvNMetaNumber of nodes208433832101Number of edges348454013266Ave. degrees3.323.183.1Ave deg of top25%8.097.827.56size of the major component191121552025% of IPA focus genes71%76%75%\nSignificant hubs (inter)\nMUC1CSF2RBDSG3MARK2DKK2HLA-DQB2CYP1A1NCF4TAF11CYP3A5NEDD9RHOGPRKCB1RUNX1BLVRB\nSignificant hubs (intra)\nTNFTGFB1TNCTGFB1MYCPI3MYCIFNGPLAUTP53NFkBFN1NFkBTP53MMP1Consensus of the enriched canonical signaling pathwaysTop 10 enriched canonical pathways resulted from the IPA analysis using the verified gene lists were quite different from those using the full gene lists. The −log(p-values) of the canonical pathways analyzed with the verified genes list were more significant than those with the full gene lists. Figure 2 demonstrated the percentage of the up- and down-regulated genes in each pathway and the −log (p-value) of each pathway was plotted in decreasing orders. The embedded table in Figure 2 showed eight canonical pathways in consensus across three progressive stages. Antigen presentation, calcium signaling and integrin signaling pathways were highlighted as the top ranked.Enriched antigen presentation and integrin signaling pathwaysWe sought to explore the global HNSCC transcriptome by looking into the enriched antigen presentation and integrin signaling pathways (Figure 3). Emergent view of cancers as an equilibrium state of the adaptive immunity confirmed the long obscuring roles of immunoediting in tumorigenesis and metastasis [20]. In the sub-networks of the antigen presentation pathway, we identified three components, representing three major complexes–proteasome, MHC class I, and MHC class II. Significant repression of the transcriptional profiles in Meta was noted, particularly HLA-G in MHC I and HLA-DRB1 in MHC II. CALR, the endoplasmic reticulum-residing chaperone, calreticulin, was found differentially expressed in Pre and Meta; and presented itself as the hub connecting MHC I, the complement system, and THBS1.Network-based approach to address the complex mechanisms underlying the integrin adhesome has been sought out by researchers [21]. In the integrin signaling networks of the HNSCC transcriptome, we identified several repressed inter-modular hubs: ACTN2, CAPN3 and TTN in Pre and TvN; and ILK, RHO-G and VCL in Meta. GRB2, ITGA5, ITGB6, ITGB7 and MAPK8 were distinguished as topologically significant hubs in the TvN interacting networks. Table S2 provided details of the topologically significant genes in the enriched antigen presentation and integrin signaling networks.Invasiveness of HNSCC implied from the disruption of integrin signaling pathwaysGaggioli and colleagues [1] established a new model to visualize the collective invasion of co-cultured stromal fibroblasts and oral carcinoma cells (SCC12). ITGA3, ITGA5, and the RhoGTPases were required in the force-mediated matrix remodeling, by which the leading stromal fibroblasts were able to generate tracks to support SCC invasion. To exploit the biological significance of the interacting networks in the enriched pathways, we compared the topological informational scores of the integrin family with the percentage of matrix contraction due to knockdown of integrins by siRNA in stromal fibroblasts (Table 4). With regard to eight integrins, ITGA1, ITGA2, ITGA3, ITGA5, ITGAV, ITGA6, ITGB1 and ITGB4, the correlation between the topological informational scores in TvN and the matrix contraction after the knockdown was −0.867 (Pearson correlation test, p-value = 0.005). The informational scores derived from the topology of interacting networks were developed to estimate the perturbed pathological effects, i.e. blocked signaling information. Not all of the integrins were differentially expressed across three stages; hence, we could not correlate the estimated pathological effects beyond the TvN networks. Nonetheless, based on the strong association between the estimated biological significance and the experimental findings, we are convinced that network-based meta-analyses of the evolving HNSCC transcriptome could indeed recapitulate the underlying nature of disease progression.10.1371/journal.pone.0003215.t004Table 4Functional estimates of integrins in the integrin signaling networks.GenegeneIDchrsiRNAfold.prefold.tvnfold.metainfo.score.tvn*\nITGA136725q11.229.600.2061900ITGA236735q23-q3128.901−0.529050ITGA3367517q21.336.100.190460.2771820ITGA5367812q11-q136.600.56625026.3ITGAV36852q31-q3221.800.1523300ITGA636552q31.115.80.32950.256940.546360ITGB1368810p11.222.700.1563804.5ITGB4369117q252800.16508−0.316860Functional estimates of integrins in the integrin signaling networks of the HNSCC transcriptome were highly correlated to the percentage of matrix contraction due to knockdown of integrins with siRNA in stromal fibroblasts, which in turn should promote the invasion of the co-cultured oral cancer cells, SCC12. The percentages of matrix contraction due to siRNA depletion were formerly reported by Gaggioli and colleagues [1]. The higher the informational score, i.e. the lower percentage of matrix contraction due to knockdown of siRNA, indicated a more significant role in the invasion process.Pearson correlation coefficient = −0.86 P-value = 0.005.*Informational scores were derived from the topological analysis of the integrin signaling networks of the HNSCC transcriptome in TvN.Genome-wide probabilities of reporting differential gene activities in the evolving HNSCC transcriptomeIn Figure 4, we plotted the standardized accumulative probabilities (PBs) of reporting differentially expressed genes within each chromosomal segment for the HNSCC transcriptome. Several significantly reported regions were identified: 1p36, 1q21, 5q31, 6p21, 9q34, 11p15, 11q13, 12p13, 12q13, 16p13, 17q21, 19p13, 19q13 and 22q13. Hot-spot loci, for instances, 1q21, where resided S100As, CD48, and ECM1, as well as 6p21, 19p13 and 19q13 were highlighted. It was of interest to know if genes co-localized in a hot-spot would interact with those within another hot-spot. In the antigen presentation pathway, MHC complex, TAP1 and TNF were located in 6p21, whereas another interacting gene, CALR, was in the other hot-spot of 19p13. We compared hot-spots of differential gene activities to those of genomic alterations reported by Weber and colleagues [2]. Correspondingly, 19p13 region was demonstrated to have loss of heterozygosity in both the stroma and the epithelium compartments of HNSCC; and 19q13–a stroma-specific locus - was correlated to the clinical nodal status. Table S3 listed highly reported genes within the hot-spots of 6p21, 19p13, and 19q13. Bounded fold changes of the differential gene expression profiles within 6p21 (167 genes) and 19q13 (189 genes) were illustrated across three progressive stages in Figure 4 c,d. In 6p21.3, HIST1H4s were found repressed in Pre and TvN; HIST1H2s induced in TvN; and HIST1H3s repressed in TvN.10.1371/journal.pone.0003215.g004Figure 4Genome-wide probabilities of the evolving HNSCC transcriptome.a. We computed the accumulative probabilities of reporting differentially expressed genes within each cytoband along the chromosomal coordinates. Hot-spots of differential gene activities, 1p21, 6p21, 19p13 and 19q13, were highlighted. The standardized accumulative probability for each cytoband was plotted across the genome. The color schemes represented three progressive stages of the HNSCC transcriptome–red circles: Meta; blue squares: TvN; and green triangles: Pre. b. Co-localization between hot-spots of differential gene activities and previously implicated regions of genomic alterations by Weber et al. [2]. c–d. Differential gene expression profiles–bounded fold changes - of 167 genes in 6p21 and 189 genes in 19q13 were plotted along the chromosomal coordinates for the Pre, TvN and Meta stages, respectively.DiscussionIn order to fully utilize the powerful gene expression profiling, challenges remained in data integration and in gaining meaningful insights. The purpose of this meta-analysis aimed at investigating the transcriptional profiles of HNSCC via a systems-level and knowledge-based network approach by means of synthesizing previous research efforts. We showed that gene-gene interplays in the interacting networks, which were quantitatively analyzed with topological informational scores and qualitatively visualized with bounded fold changes, illuminated the underlying biological events in three progressive stages of HNSCC. To date, we conducted for the first time a genome-wide meta-analysis in the context of knowledge-based networks and systematic reviews. We took the first step to unravel the pathophysiology of HNSCC via a systems-biology approach. Moreover, we provided a platform for the HNSCC research community to make strides in advancing our knowledge and future progress.We demonstrated that the perturbation of genes in the interacting networks could be estimated by the topological properties. In the enriched antigen presentation and integrin signaling pathways, we recognized significant roles of CALR, TAP1, HLA-DQB2, PTEN, HRAS, RHOA, ITGA3 and IT GA5 from the topological scorings and the differential gene activities in the evolving HNSCC transcriptome. Remarkably, the estimated informational scores derived from the integrin signaling networks not only supported previous findings of Gaggioli et al. [1], but also extended the functional implication in vivo. Most of the differentially expressed genes in the antigen presentation and integrin signaling pathways fell into hot-spot regions of 6p21 and 17q21. Moreover, several highlighted hot-spots of differential gene activities, such as 19p13 and 19q13, co-localized to previous implicated regions of genomic alterations by Weber et al. [2]. Thus, we supported and further extended findings from genomic instabilities to genome-wide differential gene expression profiles. Disrupted genes exclusively found in each stage of the HNSCC trascriptome might serve as candidate targets for future cancer prevention, targeted treatment, or drug discovery. Altogether, we hope the effort in putting systematic information into perspective will lead to the medical advancement of HNSCC.It is of importance to acknowledge that the approach taken in this meta-analysis, including converting identifiers, standardizing bounded fold changes, and analyzing the topology of knowledge-based networks, are innovative but primitive. Therefore, further experimental efforts to consolidate the demonstrated findings are crucial. Nonetheless, applying a standard template for each study, so that the subsequent meta-analyses could be feasible, was indeed painstaking. Currently, we do not have a standard guideline to report genome-wide experiments in functional context. As ‘gene’ has become a vague definition and new ‘genon’ concept been proposed [22], the work presented here might bring about the initiative to come up with a standard functional format in reporting genome-wide experiments for future systematic integration. Furthermore, the limited access to most of the datasets deserves the HNSCC research community to address issues in data-sharing and public access. Collectively, the integrative transcriptome, which orchestrated differential gene expression profiles, functional annotations, chromosomal coordinates, knowledge-based interactome, and enriched signaling pathways, elucidated the evolving nature of HNSCC. As benefits of conducting systems-level meta-reviews become clear while future research advances, we hope to see a more common practice applying the same strategy in other research areas.Supporting InformationTable S1Included studies of microarray based differential gene expression profiles of HNSCC.(0.18 MB PDF)Click here for additional data file.Table S2Topologically significant genes in enriched canonical pathways.(0.06 MB PDF)Click here for additional data file.Table S3Most frequently reported genes in 6p21, 19p13, and 19q13.(0.09 MB PDF)Click here for additional data file.Text S1Details of the PubMed Query, compiled format for each study, and details of the anatomical-site-specific genes in TvN.(0.11 MB PDF)Click here for additional data file.Figure S1Detailed Figure of the Integrin Signaling Networks in Pre.(0.26 MB PDF)Click here for additional data file.Figure S2Detailed Figure of the Integrin Signaling Networks in TvN.(0.38 MB PDF)Click here for additional data file.Figure S3Detailed Figure of the Integrin Signaling Networks in Meta.(0.24 MB PDF)Click here for additional data file.Figure S4Detailed Figure of the Merged Integrin Signaling Networks, with node coloring of the differential gene expression profiles of the Meta stage.(0.56 MB PDF)Click here for additional data file.Figure S5Detailed Figure of the Merge Networks of the Antigen Presentation Pathway, with node coloring of the differential gene expression profiles of the Meta stage.(0.16 MB PDF)Click here for additional data file.\n\nREFERENCES:\n1. 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+    "id": "PMC2533118",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2533118\nAUTHORS: Jie Zhong, Michael Rist, Leanne Cooper, Corey Smith, Rajiv Khanna\n\nABSTRACT:\nBased on the life-time cost to the health care system, the Institute of Medicine has assigned the highest priority for a vaccine to control human cytomegalovirus (HCMV) disease in transplant patients and new born babies. In spite of numerous attempts successful licensure of a HCMV vaccine formulation remains elusive. Here we have developed a novel chimeric vaccine strategy based on a replication-deficient adenovirus which encodes the extracellular domain of gB protein and multiple HLA class I & II-restricted CTL epitopes from HCMV as a contiguous polypeptide. Immunisation with this chimeric vaccine consistently generated strong HCMV-specific CD8+ and CD4+ T-cells which co-expressed IFN-γ and TNF-α, while the humoral response induced by this vaccine showed strong virus neutralizing capacity. More importantly, immunization with adenoviral chimeric vaccine also afforded protection against challenge with recombinant vaccinia virus encoding HCMV antigens and this protection was associated with the induction of a pluripotent antigen-specific cellular and antibody response. Furthermore, in vitro stimulation with this adenoviral chimeric vaccine rapidly expanded multiple antigen-specific human CD8+ and CD4+ T-cells from healthy virus carriers. These studies demonstrate that the adenovirus chimeric HCMV vaccine provides an excellent platform for reconstituting protective immunity to prevent HCMV diseases in different clinical settings.\n\nBODY:\nIntroductionHuman cytomegalovirus (HCMV) is a classic example of a group of herpes viruses, which is found universally throughout all geographic locations and socioeconomic groups, and infects 50% of adults in developed countries [1]. Although HCMV does not cause clinical disease in immunocompetent individuals except as a mononucleosis-like illness which is observed in a small number of infected individuals, HCMV infection is important to the following three high-risk groups: 1) unborn babies with an immature immune system, 2) people who work with children, and 3) immunocompromised people such as organ transplant patients and HIV-infected individuals) [1]. Epidemiological studies have shown that 15%–30% of unborn babies who acquire congenital HCMV infection display a variable pattern of pathological sequelae within the first few years of life that may include hearing loss, vision impairment and mental retardation [2]. It has been estimated that in the US alone, each year 8000 newborns have health problems as a results of congenital HCMV infection, with each child costing the US health care system more than $300,000 [3]. Based on the cost and human suffering that would be relieved by reducing the disease burden associated with HCMV infection, the development of a vaccine to prevent HCMV infection or disease was assigned the highest priority, together with vaccines for HIV, TB and Malaria, by the Institute of Medicine (USA) in 1999 [4].It is now well documented that both humoral and cellular (including CD4+ T cells and CD8+ T cells) immune responses play an important role in the control of HCMV infection and disease [1], [5]. Therefore a formulation based on viral antigens that activate both humoral and cellular immunity is crucial for a successful HCMV vaccine [6], [7]. During the last 30 years, various strategies, including whole virus, subunit vaccines based on recombinant gB protein, vector vaccines expressing immunodominant antigens (gB protein, pp65 and/or IE-1 protein), DNA vaccine and dense bodies have been developed, and some of these formulations have shown encouraging results in preclinical studies and can even induce HCMV-specific immune responses in some clinical studies [8], [9], [10], [11]. However, none of these vaccines have shown convincing clinical efficacy in the control of HCMV infection or disease, and a clinically licensed HCMV vaccine is still not available.In recent years, increasing evidence has shown that HCMV-specific immune responses are not restricted to gB, pp65 and IE-1 antigens as previously understood, but are directed towards more than 70% of the HCMV reading frames [12], [13], [14], [15]. Therefore, a vaccine which can induce a broad repertoire of HCMV-specific immune responses in different ethnic populations is likely to provide more effective protection against virus-associated pathogenesis. To achieve this goal, we have designed a novel chimeric vaccine based on a replication deficient adenovirus which encodes 46 HCMV T cell epitopes from 8 different HCMV antigens, restricted through multiple HLA class I and Class II alleles, as a polyepitope [16]. This polyepitope was covalently linked to a truncated form of HCMV-encoded gB antigen which allowed the expression of the HCMV polyepitope and gB proteins as a single fusion protein. Pre-clinical evaluation of this recombinant polyepitope vaccine in HLA A2 transgenic mice (referred to as HHD-2) and humans showed that this formulation is capable of inducing pluripotent cellular and humoral immunity in vivo and also readily recalls and expands HCMV-specific CD8+ and CD4+ T cells.ResultsImmunisation of HHD-2 mice with Ad-CMVpoly and/or Ad-gB vaccine induces multiple antigen-specific cellular and humoral immunityRecent studies on the immune regulation of HCMV in healthy virus carriers and transplant patients have clearly indicated that long-term protection from viral pathogenesis is critically dependent on the induction of cellular immunity which is directed towards multiple viral antigens expressed during different stages of HCMV infection [12], [13], [14], [15], [17]. In the first set of experiments we specifically designed our vaccine strategy to induce a cellular immune response against multiple antigens of HCMV using the polyepitope technology [18]. HLA A2 transgenic mice (referred to as HHD-2) were immunised (intramuscularly) with an adenoviral vector encoding 46 HLA class I and II-restricted T cell epitopes as a polyepitope (referred to as Ad-CMVpoly; 7.5×108 pfu/mouse; Figure 1 and Table 1). Ten days after immunisation, ex vivo T cell reactivity to the HLA A2-restricted peptide epitopes (pooled; see Table 1) was assessed by ELISPOT technology. It is important to mention here that the virus dosage for vaccination was selected based on our preliminary studies where a range of varying doses were investigated (data not shown). Splenocytes were used as responder cells for the detection of epitope-specific T cells. Data presented in Figure 2A shows that we consistently observed a strong HCMV epitope-specific T cell response following Ad-CMVpoly vaccination. Analysis of T cell responses to the individual epitopes within the polyepitope sequence indicated that during primary immunisation the dominant T cell response was directed towards the VLE epitope derived from IE-1 antigen, although subdominant responses towards other HLA A2-restricted epitopes NLV (pp65), RIF (pp65), VLA (IE-1), IIY (IE-2) AVG (gB) was also detected (Figure 2B).10.1371/journal.pone.0003256.g001Figure 1Schematic representation of the construction of a recombinant adenovirus that expresses a synthetic DNA encoding for a polyepitope protein which contains 46 HCMV T-cell epitopes (see box and Table 1).Each of the alternate epitope sequences are shown in bold letters. The DNA sequence encoding this polyepitope protein was constructed using overlapping epitope sequence specific primers (referred to as CMV1 to CMV20) as described in the “Material and Methods” section. This synthetic insert was first cloned into a pBluescript II KS+ phagemid, prior to cloning into the pShuttle vector. After amplification in E.coli, the expression cassette from pShuttle was excised and ligated into the Ad5F35 expression vector. Following linearization of the DNA using Pac I restriction enzyme, the recombinant Ad5F35 vector was packaged into infectious adenovirus by transfecting HEK 293 cells, and recombinant adenovirus (referred to as Ad-CMVpoly) was harvested from transfected cells by repeated freeze-thawing cycles.10.1371/journal.pone.0003256.g002Figure 2HCMV epitope-specific T cell response following primary and secondary immunisation with Ad-CMVpoly vaccine.Two different groups of HHD-2 mice were immunised intramuscularly with Ad-CMVpoly (7.5×108 PFU/mouse). A & B, Following primary immunisation, animals were sacrificed 10 days post immunisation and HCMV epitope-specific reactivity was assessed in the splenocytes by ELISPOT assays as described in the “Material and Methods” section. The epitopes tested for T cell reactivity were VLE (IE-1), NLV (pp65), RIF (pp65), VLA (IE-1), IIY (IE-2) AVG (gB). C & D, For immunological analysis following secondary immunisation, animals were given booster immunisation (7.5×108 PFU/mouse) 100 days after primary immunisation and then sacrificed 10 days post secondary immunisation. HCMV epitope-specific reactivity was assessed as described above. A & C shows ELISPOT data based on the pooled HLA A2-restricted HCMV epitopes, while B & D shows relative T cell responses to individual epitopes. The results are expressed as Mean±SE of spot forming cells (SFC) per 106 splenocytes from four individually tested mice. E, Anti-adenovirus antibody titre induced by immunisation with Ad-CMVpoly. Serum samples were collected at different time points after immunisation and anti-adenovirus titres were evaluated by ELISA as described in the “Material and Methods” section. All statistical analyses were conducted using GraphPad Prism 4 software.10.1371/journal.pone.0003256.t001Table 1List of HLA-restricted HCMV T cell epitopes included in the Ad-CMVpoly and Ad-gBCMVpoly.Epitope order numberEpitope SequencesHLA restrictionHCMV AntigensParent protein (UL)Amino acid locationAbbreviated codeReference1VTEHDTLLYA1pp50UL44245–253VTE\n[13]\n2KPGKISHIMLDVAB35/DR3pp65UL83283–295KPG\n[13]\n3NTDFRVLELA1gBUL55657–665NTD\n[13]\n4VLEETSVMLA2IE1UL123316–324VLE\n[13]\n5NLVPMVATVA2pp65UL83495–503NLV\n[55]\n6RIFAELEGVA2pp65UL83522–530RIF\n[13]\n7IIYTRNHEVA2IE2UL122244–251IIY\n[16]\n8CVETMCNEYA1IE1UL123279–287CVE\n[13]\n9VLAELVKQIA2IE1UL12381–89VLA\n[13]\n10AVGGAVASVA2gBUL55731–739AVG\n[13]\n11TVRSHCVSKA3pp50UL4452–60TVR\n[13]\n12IMREFNSYKA3gBUL55682–690IMR\n[13]\n13GPISHGHVLKA11pp65UL8316–24GPI\n[56], [57]\n14AYAQKIFKILA23/A24pp65?UL83248–257AYA\n[13]\n15QYDPVAALFA24pp65UL83341–349QYD\n[13]\n16YVKVYLESFA26pp65UL83223–231YVK\n[13]\n17DIYRIFAELA26pp65UL83519–527DIY\n[13]\n18VFETSGGLVVA29gBUL55420–429VFE\n[56], [57]\n19KARDHLAVLB7pp150UL32101–109KARD\n[13]\n20KARAKKDELB7/B8IE1UL123192–200KARA\n[13]\n21TRATKMQVIB57/B58/Cw6pp65UL83211–219TRA\n[13]\n22HELLVLVKKAQLDR11gHUL75276–287HEL\n[58]\n23DDYSNTHSTRYVDR7gBUL55216–227DDY\n[58]\n24QIKVRVDMVB8IE1UL12388–96QIK\n[13]\n25RRRHRQDALB8/B27pp65UL83539–547RRR\n[13]\n26ARVYEIKCRB27DNAseUL98274–282ARV\n[13]\n27NVRRSWEELB7pp150UL32212–220NVR\n[13]\n28CPSQEPMSIYVYB35pp65UL83103–114CPS\n[16]\n29QARLTVSGLB7pp65UL83158–166QAR\n[13]\n30ELKRKMMYMB8IE1UL123199–207ELK\n[13]\n31IPSINVHHYB35pp65UL83123–131IPS\n[59]\n32FEQPTETPPB41IE2UL122381–389FEQ\n[16]\n33YAYIYTTYLB41gBUL55153–161YAY\n[16]\n34QEFFWDANDIYB44/DRw52pp65UL83511–521QEF\n[13]\n35YEQHKITSYB44pp50UL44372–380YEQ\n[13]\n36QEPMSIYVYB44pp65UL83106–114QEP\n[13]\n37SEHPTFTSQYB44pp65UL83364–373SEH\n[13]\n38QAIRETVELB57/B58pp65UL83331–339QAI\n[16]\n39CEDVPSGKLB40/60pp65UL83232–240CED\n[60]\n40KMQVIGDQYB40/60pp65UL83215–223KMQ\n[60]\n41ATVQGQNLKA11pp65UL83501–509ATV\n[60]\n42HERNGFTVLB40/60pp65UL83267–275HER\n[60]\n43DALPGPCIB51pp65UL83546–552DAL\n[60]\n44VYALPLKMLA24pp65UL83113–121VYA\n[61]\n45PTFTSQYRIQGKLB38/DR11pp65UL83367–379PTF\n[55], [62]\n46QMWQARLTVB52pp65UL83155–163QMW\n[30]\nRecent studies have raised some concerns on the use of adenoviral vectors in humans as the pre-existing immunity to adenovirus may compromise the efficacy of these vaccine formulations [19], [20], [21]. To explore this issue, Ad-CMVpoly immunized HHD-2 mice were rested for one or three months and then immunized with the Ad-CMVpoly vaccine (7.5×108 pfu/mouse). Although secondary immunization of mice after one month of vaccination showed very minimal increase in the T cell response (data not shown), a 3–5 fold increase in the T cell response was observed in HHD-2 mice vaccinated after three months of their primary vaccination (Figure 2C). More importantly, following secondary immunization, a small but significant increase in the subdominant responses was observed in some animals, while the T cell response towards VLE epitope remained the most dominant component of overall response (Figure 2D). It was interesting to note that the hierarchy of these T cell responses in HHD-2 mice was very similar to that observed in HLA A2-positive healthy virus carriers [12], [13], [14]. This observation was co-incident with the dramatic decline of antibodies against adenovirus vector 2 to 3 months after immunisation with Ad-CMVpoly (Figure 2E).Combination of Ad-CMVpoly and Ad-gB induces long lasting memory cellular and humoral immune responsesIt is now firmly established that although T cell responses play an important role in controlling persistent HCMV infection, humoral immune responses also contribute significantly in controlling primary HCMV infection and as well reduce viral load by neutralizing the extra-cellular virus [22], [23]. To ensure that our vaccine strategy can induce both cellular and humoral immune responses, we immunised HHD-2 mice with the mixture of Ad-gB (7.5×108 pfu/mouse) and Ad-CMVpoly (7.5×108 pfu/mouse), and anti-HCMV specific cellular and humoral immune responses in immunised animals were evaluated at different time point by ELISPOT and ELISA respectively. Data presented in Figure 3A & B shows that this vaccination strategy induced both CD8+ T cells and gB-specific antibody responses. The levels of CD8+ T cells induced by this co-immunisation strategy were comparable to those seen with Ad-CMVpoly alone and were detectable at reasonably high levels on day 75 post-immunisation (Figure 3A). The levels of gB-specific antibody responses were maintained at high levels by day 75 post-immunisation, although a small reduction was observed when compared to the levels observed on days 10 and 25 respectively. In contrast, the antibody response showed significant increase in the virus neutralization capacity by day 75 post-immunisation (Figure 3C) which was co-incident with the antibody avidity maturation (Figure 3D). It is important to mention here that this increase in neutralization capacity was not due to antibody isotype switching (Figure 3E), which was consist with previous studies [24]. These observations suggested that co-delivery of HCMV polyepitope and gB vaccine can induce long lasting memory T cells immunity and antibody responses.10.1371/journal.pone.0003256.g003Figure 3HCMV-specific effector and memory cellular and humoral immune responses following immunisation with a mixture of Ad-CMVpoly and Ad-gB vaccines.\nA, HCMV-specific CD8+ T cell responses following immunisation with Ad-CMVpoly and Ad-gB. These T cell responses were assessed using ELISPOT assays on day 10, 25 and 75 post immunisation. The results are expressed as Mean±SE of spot forming cells (SFC) per 106 splenocytes. B, gB-specific antibody responses in serum samples from immunised mice on days 10, 25 and 75. Serum samples on day 0 were collected before the immunisation. C, Virus neutralizing capacity of antibody responses induced in HHD-2 mice immunised with Ad-CMVpoly and Ad-gB. Serum samples from these mice were pre-incubated with HCMV virus Ad169 and then these virus preps were used to infect MRC-5. Following overnight incubation virus infectivity was assessed using IE-1/IE-2 expression as outlined in the “Material and Methods” section. D, Avidity maturation of gB-specific antibody responses in Ad-CMVpoly and Ad-gB immunised mice. E, Immunoglobulin subclass analysis of gB-specific antibody responses in HHD-2 vaccinated mice. Serum samples were collected from three different groups of mice on days 10, 25 and 75 post-immunisation. A minimum of five mice from each group were assessed for HCMV epitope-specific T cell reactivity and humoral immune responses. All statistical analyses were conducted using GraphPad Prism 4 software.Covalent linking of HCMV polyepitope with extracellular gB induces pluripotent T cell and antibody responsesAlthough co-immunisation with Ad-gB and Ad-CMVpoly induced both humoral and cellular immune responses against HCMV, delivery of this formulation in a human setting may face significant regulatory constraints. To overcome this potential limitation, we constructed another recombinant adenovirus expressing the extracellular domain of gB and HCMV polyepitope as a single polypeptide (referred as Ad-gBCMVpoly). HHD-2 mice were immunised with the Ad-gBCMVpoly vaccine (7.5×108 pfu/mouse) and both humoral and cellular immune responses were evaluated at the indicated time points. Data presented in Figure 4A shows that immunisation with Ad-gBCMVpoly vaccine induced a long-tem memory CD8+ T cell response towards the HLA A2-restricted epitopes from HCMV. Furthermore these animals also showed strong gB-specific antibody response and similar to the data presented in Figure 3B, the levels of gB-specific antibody dropped by day 75 post immunisation (Figure 4B). A significant increase in the neutralizing activity of the antibody response was observed (Figure 4C), which was co-incident with avidity maturation (Figure 4D). On the other hand, there was no antibody isotype switching at different time point after immunisation (Figure 4E). These observations clearly demonstrated that covalent linking of the gB with the polyepitope sequence does not impair the immunogenicity of each of the components of the vaccine.10.1371/journal.pone.0003256.g004Figure 4HCMV-specific effector and memory humoral and cellular immune responses following immunisation with Ad-gBCMVpoly vaccine.\nA, HCMV-specific CD8+ T cell responses following immunisation with Ad-gBCMVpoly. These T cell responses were assessed using ELISPOT assays on day 10, 25 and 75 post immunisation. The results are expressed as Mean±SE of spot forming cells (SFC) per 106 splenocytes. B, gB-specific antibody responses in serum samples from immunised mice on days 10, 25 and 75. Serum samples on day 0 were collected before the immunisation. C, Virus neutralizing capacity of antibody responses induced following immunisation with Ad-gBCMVpoly. Serum samples from these mice were pre-incubated with HCMV virus Ad169 and then these virus preps were used to infect MRC-5. Following overnight incubation virus infectivity was assessed using IE-1/IE-2 expression as outlined in the “Material and Methods” section. D, Avidity maturation of gB-specific antibody responses in Ad-gBCMVpoly immunised mice. E, Immunoglobulin subclass analysis of gB-specific antibody responses in HHD-2 vaccinated mice. Serum samples were collected from three different groups of mice on days 10, 25 and 75 post-immunisation. A minimum of five mice from each group were assessed for HCMV epitope-specific T cell reactivity and humoral immune responses. All statistical analyses were conducted using GraphPad Prism 4 software.To further characterize the T cell responses induced by Ad-gBCMVpoly vaccine, we next assessed whether immunisation with Ad-gBCMVpoly result in the differentiation of antigen-specific T cells into fully functional effectors. A number of recent studies have demonstrated that the production of TNF-α in addition to IFN-γ by T-cells is a characteristic of greater differentiation and can enhance protection against infectious pathogens [25], [26], and the translocation of CD107a from intracellular lysosomal and endosomal compartments to the surface of CD8+ T cells is a positive marker of degranulation, a requisite process of perforin-granzyme mediated killing function of CTLs [27], [28]. We assessed the level of TNF-α and/or CD107a expression by IFN-γ expressing CD8+ T-cells using intracellular cytokine assays. Data presented in Figure 5A–B shows that following ex vivo stimulation with HCMV epitopes, CD8+ T cells from these mice showed strong IFN-γ expression and a large proportion of these T cells also expressed TNF-α and/or CD107a. Furthermore, after in vitro stimulation with individual HCMV peptides, these HCMV peptide-specific CD8+ T cells could be expanded and expressed both IFN-γ and TNF-α (Figure 5C–E).10.1371/journal.pone.0003256.g005Figure 5Cytokine expression by HCMV-specific CD8+ T cells from Ad-gBCMVpoly immunised HHD-2 mice.\nA & B, Ex vivo expression of IFN-γ, TNF-α and CD107a by antigen-specific CD8+ T-cells from Ad-gBCMVPpoly vaccinated mice 10 days post-vaccination. Splenocytes were prepared from 3 individual HHD-2 mice 10 days post-vaccination and cultured with individual HCMV peptides overnight. Anti-CD107a antibody and Brefeldin A was added during the last 6 and 5 hours incubation respectively, followed by T cell surface marker and intracellular cytokine staining. Data represent the percentage of IFN-γ expressing CD8+ T cells (A) and percentage of single, double or triple markers expressing cells among IFN-γ expressing CD8+ T cells (B). C–E, Expression of IFN-γ and/or TNF-α by in vitro expanded antigen-specific CD8+ T-cells from Ad-gBCMVPpoly vaccinated mice 10 days post-vaccination. Splenocytes pooled from three immunised mice were first stimulated with individual HCMV peptide epitope-pulsed splenocytes for 2 weeks in the presence of recombinant mouse IL-2 at the concentration of 10 IU/ml, then cultured with MRC-5 cells pulsed with corresponding HCMV peptide epitope overnight for intracellular cytokine assay. The HCMV peptide epitopes tested here were VLE (IE-1), NLV (pp65), RIF (pp65), VLA (IE-1) at the concentration of 1 µg/ml. Data represent the percentage of IFN-γ (C), TNF-α (D) and IFN-γ & TNF-α (E) expressing CD8+ T-cells. ** (p<0.005) and * (p<0.05) show statistically significant difference between indicated CMV peptide epitopes and control epitope (A). Data from one out three experiments with similar results was shown in C–E. All statistical analyses were conducted using GraphPad Prism 4 software.Protection against quasi-virus challenge following immunisation with Ad-gBCMVpolyHaving firmly established the immunogenicity of Ad-gBCMVpoly vaccine, the next set of experiments was designed to determine protective efficacy of this vaccine. Due to the species restriction, we challenge immunised HHD-2 mice with recombinant vaccinia encoding HCMV antigens (gB and IE-1) to evaluate the protective efficiency of the Ad-gBCMVpoly vaccine. Data presented in Figure 6A shows that HLA A2 mice immunised with Ad-gBCMVpoly vaccine showed significant reduction in the virus load following challenge with Vacc.gB and Vacc.IE-1. This reduction in the virus load was highly antigen-specific as the vaccinated or naïve animals challenged with Vacc.TK- or Vacc.gB, Vacc.IE-1 respectively showed minimal reduction in the viral load. Although Ad-gBCMVpoly immunized mice showed better protection against Vacc.gB when compared to Vacc.IE-1 (Figure 6A), this better protection was not due to anti-gB antibodies (Figure 6B) as Vacc.gB was not neutralized by serum from immunized animals (data not shown), but due to gB-specific CD4+ T cell responses (Figure 6C). Nevertheless, the anti-gB humoral response should play an important role in human as it induces HCMV neutralizing antibodies. As expected, the reduction in the Vacc.IE-1 virus load in Ad-gBCMVpoly immunised mice was co-incident with the induction of VLE-specific CD8+ T cell responses (Figure 6D). It is important to note that Ad-gBCMVpoly immunised mice challenged with Vacc.gB or Vacc.IE-1 showed significantly higher humoral and T cell responses respectively when compared to mice challenged with Vacc.TK-. We also assessed the level of TNF-α expression by IFN-γ expressing CD4+ and CD8+ T-cells using intracellular cytokine assays. Data presented in Figure 6E shows that following stimulation with gB protein or HCMV IE-1 epitope, a large proportion of CD4+ and CD8+ T cells from these mice showed strong co-expression of IFN-γ and TNF-α.10.1371/journal.pone.0003256.g006Figure 6Ad-gBCMVpoly induced protection against challenge with recombinant vaccinia expressing gB or IE-1 protein.HHD-2 mice were immunised with Ad-gbCMVpoly vaccine and 21 days following vaccination these mice were challenged (intraperitoneal) with recombinant vaccinia encoding gB (Vacc.gB), IE1 protein (Vacc.IE-1) or control vaccinia (Vacc.TK−) at 107 pfu virus/mouse. Ovaries, splenocytes and peripheral blood samples were collected four days later and used for assessing viral load, antigen-specific T cell response and gB-specific antibody response. A, Virus titres in the ovaries of Ad-gBCMVpoly immunised or naïve HHD-2 mice challenged with Vacc.IE-1, Vacc.gB or Vacc.TK−. B, gB-specific antibody response in Ad-gBCMVpoly immunised or naïve HHD-2 mice challenged with Vacc.gB or Vacc.TK−. C,\nEx vivo gB-specific CD3+CD4+ T cell response in Ad-gBCMVpoly immunised or naïve HHD-2 mice challenged with Vacc.gB or Vacc.TK−. Splenocytes from these mice were stimulated with recombinant gB protein (40 µg/ml) overnight and then assessed for IFN-γ production using intracellular cytokine assay. D,\nEx vivo IE-1-specific CD3+CD8+ T cell response in Ad-gBCMVpoly immunised or naïve HHD-2 mice challenged with Vacc.IE-1 or Vacc.TK−. Splenocytes from these mice were stimulated with the peptide epitope VLEETSVML (1 µg/ml) overnight and then assessed for IFN-γ production using intracellular cytokine assay. E,\nEx vivo expression of IFN-γ and/or TNF-α by antigen-specific CD8+ and CD4+T-cells from Ad-gBCMVPpoly vaccinated mice, challenged with recombinant vaccinia encoding IE-1 or gB. Splenocytes from immunised mice stimulated with either gB protein or IE-1 peptide epitope overnight for intracellular cytokine assay. Data represent the percentage of TNF-α and IFN-γ & TNF-α expressing CD4+ or CD8+ T-cells. All statistical analyses were conducted using GraphPad Prism 4 software.Expansion of multiple antigen-specific human CD8+ and CD4+ T cells following stimulation with Ad-gBCMVpolyAnother important aspect of the current study was aimed at exploring the potential efficacy of Ad-gBCMVpoly to recall memory T cell responses from healthy seropositive individuals. PBMC from healthy donors were stimulated with irradiated autologous PBMC-infected with Ad-gBCMVpoly. Following stimulation these T cells were assessed for antigen specificity using intracellular cytokine assays. Data for the gB-specific CD4+ and CD8+ T cell responses are summarised in Figure 7, while the T cell responses towards the epitopes within the polyepitope sequence are presented in Tables 2 & 3. To identify the gB-specific T cell responses we used an overlapping set of peptides based on the gB sequence from Ad169 strain of HCMV. This analysis showed that following stimulation with Ad-gBCMVpoly, more than 88% of the individuals showed expansion of gB-specific CD4+ T cells. These T cell expansions raged from 2–36% of the total CD3+ CD4+ T cells (Figure 7). CD8+ T cell responses directed towards gB epitopes were detected in 70.5% donors which ranged from 2–15% of the total CD3+ CD8+ T cells. T cells from each donor recognized multiple gB epitopes and most of the donors demonstrated a selective expansion of gB-specific CD4+ or CD8+ T cells.10.1371/journal.pone.0003256.g007Figure 7Expansion of gB-specific T cells following in vitro stimulation of human PBMC with Ad-gBCMVpoly.PBMC from a panel of healthy virus carriers (referred to as D1–D17) were co-cultured with autologous PBMC infected with Ad-gBCMVpoly (MOI: 5∶1 or 1∶1) at a responder to stimulator ratio of 2∶1. These cultures were supplemented with rIL-2 (10 U/ml) on day 3 and every 3–4 days thereafter. On day 14, these T cell cultures were tested against a panel of pooled overlapping gB peptides (20 aa long, overlapping by 10 aa) using intracellular cytokine assays. The data presented in the figure shows the percentage of gB-specific CD8+ and CD4+ T cell recovered from each donor following stimulation with Ad-gBCMVpoly.10.1371/journal.pone.0003256.t002Table 2CD8+ T cell responses in healthy virus carriers following stimulation with Ad-gBCMVpolya.HCMV EpitopesD1 (A1 A29 B8 B44)D2 (A1 A3 B7 B8)D3 (A1 A24 B8 B14)D4 (A23 A24 B27 B41)D5 (A11 A24 B35 B60)D6 (A1 A31 B8 B51)D7 (A1 A11 B8 B35)D8 (A24 A26 B15 B62)D9 (A2 A11 B13 B27)D10 (A2 B35 B57)D11 (A3 A23 B35 B44)D12 (A24 A26 B35 B38)D13 (A1 A2 B7 B57)D14 (A1 A2 B44)D15 (A31 A33 B35 B58)D16 (A1 A2 B7 B37 Cw6&7)D17 (A1 A1 B8 B8 Cw7)D18 (A2 A68 B8 B15 Cw4&7)VTE (HLA A1; pp50)+++b\n+++++++++++++++++++++++++++++KPG (HLA B35; pp65)+/−+/−++++/−+/−++++NTD (HLA A1; gB)+/−+/−+/−+/−+/−+/−+/−+/−+/−VLE (HLA A2; IE-1)+++++++++++++++/−++++NLV (HLA A2; pp65)+++++++++++/−+/−++++RIF (HLA A2 pp65)++++++++/−+/−+/−+++IIY (HLA A2 IE-2)+/−+++++++/−++++CVE (HLA A1; IE-1)+/−+/−+/−+/−+/−+++/−+/−+/−VLA (HLA A2; IE-1)+/−+++/−+/−+/−+/−AVG (HLA A2; gB)+/−++++/−+/−+++/−TVR (HLA A3; pp50)+/−+/−IMR (HLA A3; gB)+/−+/−GPI (HLA A11; pp65)+/−+/−+/−AYA (HLA A23/A24; pp65)++++++/−+++++++/−QYD (HLA A24; pp65)+/−+/−+/−++++YVK (HLA A26; pp65)++++DIY (HLA A26; pp65)+/−+/−VFE (HLA A29; gB)+/−KARD (HLA B7; pp150)+/−+/−+/−KARA (HLA B7/B8; IE-1)+/−+/−+/−+/−+/−+/−+/−+/−+/−TRA (HLA B57/B58; pp65)+++++/−+/−HEL (HLA DR11; gH)+/−DDY (HLA DR7; gB)++QIK (HLA B8; IE-1)++++++++++++++++++++++++RRR (HLA B8/B27; pp65)+/−+/−+/−+/−+/−+/−+/−+/−+/−ARV (HLA B27; pp28)+++++/−NVR (HLA B7; pp150)+/−+++/−CPS (HLA B35; pp65)+/−+++++++++++++/−+/−QAR (HLA B7; pp65)+/−+/−+/−ELR (HLA B8; IE-1)+++++++++++++++++++++++IPS (HLA B35; pp65)+++++/−+/−+++++/−+++FEQ (HLA B41; IE-2)++++YAY (HLA B41; gB)++++QEF (HLA B44; pp65)+/−+++++/−+/−YEQ(HLA B44; pp50)+++/−+/−QEP (HLA B44; pp65)+/−++++/−+/−SHE (HLA B44; pp65)+++++/−QAI (HLA B57/B58 pp65)++++CED (HLA B40/60; pp65)+/−KMQ (HLA B40/60; pp65)+/−ATV (HLA A11; pp65)+/−+/−+/−HER (HLA B40/60; pp65)+/−DAL(HLA B51; pp65)+/−VYA (HLA A24; pp65)+/−+/−+/−+++++PTF (HLA B38; pp65)+/−+/−+/−QMW (HLA B52; pp65)aT cell response was assessed by intracellular cytokine assays for the secretion of IFN-γ.bProportion of IFN-γ-secreting CD8+ T cells +/−: <1%, ++: 1–4%, +++: 5–10%, ++++: >10%.10.1371/journal.pone.0003256.t003Table 3CD4+ T cell responses in healthy virus carriers following stimulation with Ad-gBCMVpolya.HCMV EpitopesD1 (A1 A29 B8 B44)D2 (A1 A3 B7 B8)D3 (A1 A24 B8 B14)D4 (A23 A24 B27 B41)D5 (A11 A24 B35 B60)D6 (A1 A31 B8 B51)D7 (A1 A11 B8 B35)D8 (A24 A26 B15 B62)D9 (A2 A11 B13 B27)D10 (A2 B35 B57)D11 (A3 A23 B35 B44)D12 (A24 A26 B35 B38)D13 (A1 A2 B7 B57)D14 (A1 A2 B44)D15 (A31 A33 B35 B58)D16 (A1 A2 B7 B37 Cw6&7)D17 (A1 A1 B8 B8 Cw7)D18 (A2 A68 B8 B15 Cw4&7)VTE (pp50)++b\n+++++++++++/−+++++++KPG (pp65)+++/−+++/−+/−++NTD (gB)+/−+/−+++/−+/−+/−+++/−+/−VLE (IE-1)+/−+/−+/−+++/−+/−NLV (pp65)+++/−+/−+++/−++RIF (pp65)+/−+/−+/−+++/−+/−IIY (IE-2)+/−+/−+/−+/−+/−+/−CVE (IE-1)+/−+/−+++/−+/−+/−+++/−+/−VLA (IE-1)+/−+/−+/−+++/−+/−AVG (gB)+/−+/−+/−+++/−+/−TVR (pp50)+/−+/−IMR (gB)+/−+/−GPI (pp65)+/−+/−+/−AYA (pp65)+/−+/−+/−+++++/−QYD (pp65)+/−+++/−+++/−YVK (pp65)+++/−DIY (pp65)+/−+/−VFE (gB)+/−KARD (pp150)+/−+/−+/−KARA (IE-1)+/−+/−+++/−+/−+/−+/−+/−+/−TRA (pp65)+/−+/−+/−HEL (gH)+++DDY (gB)++++QIK (IE-1)++++++++++++++/−RRR (pp65)+/−+/−+/−+/−+/−+/−+/−+/−+/−ARV (pp28)+/−+/−NVR (pp150)+/−+/−+/−CPS (pp65)+/−+++/−+++/−+/−QAR (pp65)+/−+/−+/−ELR (IE-1)+++/−++++++++++/−IPS (pp65)++++/−+/−+++/−++FEQ (IE-2)++YAY (gB)++QEF (pp65)+/−++++++++YEQ(pp50)+/−++++QEP (pp65)+/−+/−++SHE (pp65)+++/−+/−QAI (pp65)+++CED (pp65)+/−KMQ (pp65)+/−ATV (pp65)+/−+/−+/−HER (pp65)+/−DAL(pp65)+/−VYA (pp65)+++/−+/−+++/−PTF (pp65)+++++/−++++QMW (pp65)aT cell response was assessed by intracellular cytokine assays for the secretion of IFN-γ.bProportion of IFN-γ-secreting CD4+ T cells +/−: <1%, ++: 1–4%, +++: 5–10%, ++++: >10%.Analysis of the T cell responses towards the epitopes within the polyepitope sequence revealed that there was a rapid expansion of CD8+ T cells following stimulation with Ad-gBCMVpoly which recognized multiple epitopes restricted through a number of HLA class I alleles (Table 2). In most cases, dominant CD8+ T cell expansions directed towards 2–3 different epitopes was observed; whilst in other donors (e.g. D9, D10 and D13) strong T cell reactivity towards more than five epitopes was observed. In vitro testing of these T cells also showed that these cells expressed high levels of CD107 and efficiently recognized HLA-matched HCMV-infected target cells (data not shown). These observations were also confirmed by ex vivo stimulating the PBMC from healthy virus carriers with Ad-gBCMVpoly. A representative data presented in Figure 8 clearly shows that ex vivo stimulation of PBMC rapidly stimulated HCMV epitope specific T cells and these cells showed strong expression of IFN-γ. Although the polyepitope sequence was predominantly based on CD8+ T cell epitopes, two previously mapped CD4+ T cell epitopes were also included in this sequence. As expected, a strong expansion of CD4+ T cells specific for these epitopes was observed, however unexpectedly, we also detected low to medium levels of expansion of CD4+ T cells which showed reactivity against HLA class I-restricted CD8+ T cell epitopes (Table 3). A careful analysis of these CD8+ T cell epitopes revealed that many of these sequences overlapped the CD4 epitopes mapped recently by other investigators [29], [30], [31].10.1371/journal.pone.0003256.g008Figure 8\nEx vivo stimulation of human PBMC with Ad-gBCMVpoly.PBMC from healthy virus carriers were co-cultured with autologous PBMC infected with Ad-gBCMVpoly (MOI: 5∶1) at a responder to stimulator ratio of 2∶1 for 6 h. These T cells were then co-stained with anti-CD3, anti-CD8, PE-labelled anti-INF-γ antibody and APC-labelled MHC-peptide multimers. A–F, Percentage of CD8+ T cells expressing INF-γ following mock stimulation or Ad-gBCMVpoly stimulation. G–L, Percentage of MHC-peptide pentamer-positive cells expressing IFN-γ following mock stimulation or Ad-gBCMVpoly stimulation. Pentamers used for each of the HCMV epitopes are indicated in G–L.DiscussionThe data presented in this study provides a highly efficient strategy for the prevention of HCMV disease in different clinical settings ranging from congenital infection to primary or reactivation of the virus in immunosuppressed adults. The importance of HCMV as the leading infectious cause of mental retardation and other abnormalities such as deafness in children has been emphasized by its categorization by the Institute of Medicine as a Level I vaccine candidate [i.e. most favourable impact–saves both money and quality-adjusted life years] [4]. Immunocompromised individuals such as transplant recipients and HIV-infected individuals with CD4 counts below 50/µl are also impacted by HCMV infection and this virus is regarded as the most important viral pathogen affecting transplantation, including both solid organ transplant and allogeneic hematopoietic stem cell transplant recipients [1], [32], [33]. Extensive studies over the last decade on the immunobiology of HCMV infection has provided detailed insight into the immune regulation of persistent HCMV infection in healthy virus carriers and individuals with HCMV-associated diseases [1]. Based on these observations, a number of attempts have been made to design a prophylactic vaccine for the control of HCMV infection. The first series of attempts focussed on the use of an attenuated form of the virus as a vaccine [6], [8], [34], [35] however, disappointing results coupled with the regulatory problems associated with the live attenuated HCMV vaccine prompted investigators to switch to the recombinant subunit approach [36], [37], [38], [39]. Although the subunit vaccine delivery systems and modalities based on HCMV encoded antigens such as gB, pp65 and IE-1 have failed to result in a licensed clinical product, interesting pre-clinical (based on animal models) and clinical data continues to accumulate demonstrating that subunit vaccination has a protective effect against congenital transmission [9], [10], [40], [41], [42].It is now firmly established that long-term latent HCMV infection is very efficiently controlled by virus-specific CD4+ and CD8+ T cells [12], [13], [14], [15], [43]. Perturbation in the regulation of T cell control often triggers reactivation of HCMV and development of HCMV-associated diseases [17], [44], [45]. The concept that a vaccine based on T cell-mediated control would be effective in controlling HCMV diseases grew out of the pioneering work conducted by Riddell and colleagues, who showed that adoptive transfer of donor-derived virus-specific T cells alone were sufficient to reduce the incidence of HCMV disease in allogeneic hematopoietic stem cell transplant recipients [46], [47]. Over the last few years there has been a series of attempts to develop a highly tailored vaccine strategy designed to induce T cell immunity against pp65 and/or IE-1 antigens or defined T cell epitopes from these antigens [38], [48], [49].While these strategies provided specificity and safety, their application at the population level are rather limited. Thus other approaches which target multiple antigens might be an advantage by providing wider coverage in different ethnic groups. Furthermore, inclusion of a virus neutralization component in the vaccine formulation has been argued by many investigators, especially in the context of congenital HCMV infection. Indeed the chimeric vaccine developed in this study induced high avidity humoral responses and cellular immunity with a single formulation and provided wider coverage through the inclusion of multiple T cell epitopes restricted through a range of HLA class I and II alleles. Our initial studies with a mixture of adenoviral vectors encoding HCMV polyepitope sequence and gB protein showed that it was possible to induce both humoral and cellular immune responses without compromising the immunogenicity of individual components of the vaccine. Taking into consideration these observations, we designed a chimeric vaccine in which the encoding sequence for the extracellular domain of gB was covalently linked with the polyepitope sequence. Extensive studies with this formulation provided further evidence that co-delivery of gB and the polyepitope as a single polypeptide was highly efficient in generating neutralizing antibodies responses and virus-specific CD8+ and CD4+ T cell responses in a murine model and healthy virus carriers. Subsequently, we employed an experimental animal model system to determine whether immunisation of HLA A2 transgenic mice with Ad-gBCMVpoly is capable of reducing infection with a recombinant vaccinia virus expressing HCMV antigens (i.e. gB and IE-1). These mice not only showed induction of a strong CD4+ and CD8+ T cell response following immunisation but also acquired strong resistance to virus infection. Interestingly, Ad-gBCMVpoly immunized showed better protection against Vacc. gB when compared to Vacc.IE-1, which suggested that gB-specific CD4+ T cell responses could also inhibit Vacc.gB virus.Another important outcome of this study was the longevity of the immune responses induced by the chimeric HCMV vaccine which is particularly critical for the vaccine designed to control congenital infection/disease where long-term memory response over multiple years would be essential. Although the studies outlined here does not allow any firm conclusions on the efficacy of the chimeric polyepitope-based vaccine in humans, it does clearly show that a formulation based on gB and HCMV T cell epitopes can be used as immunogens to induce efficient humoral and T cell responses in vivo. It is important to stress here that a polyepitope-based vaccine for HCMV has a number of advantages over the traditionally proposed vaccines, which are based on either full-length HCMV antigens or synthetic peptide epitopes. There is now convincing evidence that polyepitope proteins are extremely unstable and are rapidly degraded by the proteasome dependent pathway as a result of their limited secondary and tertiary structure [18]. The rapid degradation of these polypeptides dramatically enhances endogenous presentation of peptide epitopes through the class I and II pathway. On the other hand the full-length HCMV protein antigens are unlikely to be degraded rapidly and may also initiate various intracellular signalling events leading to the interference of presentation of epitopes from other antigens [50], [51]. Finally, the polyepitope-based vaccine is likely to overcome any potential problem of reinfection with different strains of HCMV and unique HLA types in different ethnic groups of the world.There is an emerging argument that HCMV vaccine efforts should focus on the development of formulation(s) which are designed to limit or prevent HCMV related diseases rather than to prevent infection itself [11]. This contention is supported by extensive studies in humans which revealed that although the immune responses generated during natural HCMV infection are unable to clear the latent virus, this response is sufficiently competent to keep the virus under control and restrict virus replication [13], [14], [15]. Furthermore, in immunocompromised patients such as HIV-infected individuals and transplant recipients, HCMV related pathogenesis is generally due to reactivation rather than primary infection. Considering the limited efficacy of the currently available HCMV vaccine formulations in protecting against infection in preclinical and clinical studies, we propose that a vaccine to limit or prevent HCMV related disease rather than infection itself is more realistic in the near future.Materials and MethodsConstruction of recombinant adenovirus encoding HCMV polyepitope, gB and gB-HCMV polyepitope fusion proteinThe amino acid sequence of the 46 contiguous HLA class I and class II-restricted T cell epitopes (Table 1) were translated to the nucleotide sequence using human universal codon usage. Oligonucleotides (102–107mer long) overlapping by 20 base pairs and representing the polyepitope DNA sequence, were annealed together by using Splicing by Overlap Extension and stepwise asymmetric PCR [16]. The final PCR product was cloned into pBluescript II KS+ phagemid (Agilent Technologies, Melbourne, Australia) encoded a Kozak sequence, Start methionine followed by 46 contiguous HLA class I and class II-restricted epitopes. The HCMV sequence encoding glycoprotein B (gB) was amplified from the AD169 virus stock by PCR using gene specific primers. This PCR product was designed to encode gB sequence from the alanine residue at position 31 to valine at position 700 with the deletion of the signal sequence. Following amplification the DNA was cloned into pBluescript II KS+ phagemid and confirmed by DNA sequence analysis. For the expression of the gB-HCMV polyepitope fusion protein the recombinant HCMV polyepitope insert was excised from the pBluescript II KS+ phagemid and cloned into the gB pBluescript construct.The assembly and production of the recombinant Ad5F35-based adenoviruses was completed in three stages using a highly efficient, ligation-based protocol of the Adeno-X System (CLONTECH, Palo Alto, CA) (See Figure 1). Firstly, inserts were excised from each of the constructs in pBluescript II KS+ phagemid using Xba I/Kpn I restriction enzymes and cloned into the pShuttle expression vector. Following amplification in E.coli, the expression cassette from pShuttle was excised using I-Ceu I/PI-Sec I homing enzymes and cloned into an Ad5F35 expression vector. The recombinant Ad5F35 vector was transfected into human embryonic kidney (HEK) 293 cells, and the recombinant adenoviruses (referred to as Ad-CMVpoly, Ad-gB and Ad-gBCMVpoly) were harvested from the transfected cells by successive freeze-thawing cycles.Synthesis of PeptidesPeptides, synthesized by the Merrifield solid phase method, were purchased from Chiron Mimotopes (Melbourne, Australia), dissolved in dimethyl sulphoxide, and diluted in serum-free RPMI 1640 medium for use in standard T cell assays. Purity of these peptides were tested by mass spectrometery and showed >90% purityAnimals and immunisationHLA A2 transgenic mice (referred to as HHD-2) [52], were maintained under conventional conditions the animal facility at the Queensland Institute of Medical Research. These mice are knocked out for β2 microglobulin and H-2Db and transgenic for a chimeric HLA-A2.1 with the α3 domain derived from H-2Db to allow interaction with murine CD8 and a covalently attached human β2 microglobulin. These mice were immunised with varying doses of plaque forming units (PFU) of recombinant viruses (Ad-CMVpoly, Ad-CMVgB and Ad-gBCMVpoly) and HCMV-specific humoral and cellular immune responses were evaluated at various time points. Protocols were approved by QIMR animal ethics committee.ELISpot assayThe ELISPOT assay was used to detect HLA A2-restricted HCMV epitope-specific T cells following stimulation with synthetic peptide(s) as described previously [13]. Briefly, 2×105 responding cells were incubated in triplicate with each peptide epitope (1 µg/ml) for 18 to 20 hrs in 96- well Multiscreen HA filtration plates (MAHA S4150, Millipore, Bedford, MA) coated with anti-IFN-γ monoclonal antibody (Mabtech AB, Nacka, Sweden). After incubation, the plates were extensively washed with Phosphate buffered saline with 0.5% Tween 20 and incubated with a second biotinylated anti-IFN-γ mAb followed by the addition of streptavidin conjugated alkaline phosphatase. Cytokine producing cells were detected as purple spots after a 30-min reaction with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium. Spots were counted automatically using image analysis software. T cell precursor frequencies for each peptide epitope were based on the total number of cells and the number of spot forming cells (SFC) per well (average of 3 wells). Epitope-specific spots were calculated after subtraction of the number of spots in control wells consisting of cells without added peptide (average of six wells).Intracellular Cytokine StainingSplenocytes from immunised mice or T cells from human donors were incubated for overnight at 37°C with HCMV peptide epitopes (1 µg/ml), or stimulator cells either pre-coated with HCMV peptide epitopes (1 µg/ml) or infected with recombinant vaccinia virus encoding HCMV antigens, in growth medium. Brefeldin A (BD Pharmingen, San Diego, CA) was added during the last 5 hour-incubation. For CD107a staining, anti-CD107a antibody was added one hour before the adding of Brefeldin A. These cells were then washed and incubated with PerCP-conjugated anti-CD8, FITC conjugated anti-CD4 and Allophycocyanin-conjugated anti-CD3 at 4°C for 30 mins. Cells were washed, then fixed and permeabilised with cytofix/cytoperm (BD Pharmingen) at 4°C for 20 minutes. Cells were then washed in perm/wash (BD Pharmingen), incubated with anti-IFN-γ and anti-TNF-α mAbs (BD Pharmingen) at 4°C for 30 mins, washed again with perm/wash, resuspended in PBS and analysed on a FACS Canto.Expansion of HCMV specific T-cells from healthy donors using Ad-gBCMVpolyA panel of 17 human volunteers were recruited for this study. Each volunteer was asked to sign the consent form as outlined in the institutional ethics guidelines. For the expansion of specific T-cells, peripheral blood mononuclear cells (PBMC) were co-cultured in multi-well tissue culture plates in growth medium with either PBMC (2,000 rad) infected with Ad-gBCMVpoly (MOI of 10∶1) at a responder to stimulator ratio of 2∶1. On day 3, and every 3–4 days thereafter, the cultures were supplemented with growth medium containing recombinant IL-2 (kindly donated by NIH AIDS Research & Reference Reagent Program). These T-cell cultures were assessed for HCMV epitope-specific reactivity on days 10–17.ELISA assay for anti-gB and anti-adenovirus antibodySerum anti-gB or anti-adenovirus antibody titres were evaluated by ELISA as previously described [53]. Briefly, PVL microplate 96-well plates (MP Biomedicals, Sydney, Australia) pre-coated with recombinant HCMV gB protein or adenovirus were incubated with serially diluted serum samples for 2 hours at room temperature. After washing with PBS-Tween-20 (PBST), plates were incubated with HRP-conjugated sheep anti-mouse Ig antibody (murine samples) or HRP-conjugated sheep anti-human Ig antibody (human samples) for 1 hour. These plates were washed and incubated with 3.3′, 5.5′-tetramethylbenzidine substrate solution (PanBio, Brisbane, Australia) and the OD at 450 nm was analysed using an ELISA reader. The isotypes of anti-gB antibodies in serum samples were determined by ELISA as described above using the mouse monoclonal antibody isotyping reagent kit (Sigma, IS02-1 kit, Sydney, Australia) according to the manufacturer's protocol.Antibody avidity was evaluated as previously described [54]. Briefly after incubation of plates with serum samples as described above, 5 M Urea (in PBST) was then added to half of the wells for dissociation and the other half received PBST without urea. After incubation for 30 min, plates were washed with PBST and incubated with HRP-conjugated sheep anti-mouse Ig antibody (murine samples) or HRP-conjugated sheep anti-human Ig antibody (human samples) for 1 hour and completed using the standard ELISA. The avidity indices were calculated as the ratio of the OD values with urea divided by the OD values without urea and expressed as a percentage.CMV microneutralization assayThe neutralizing activity of the anti-gB antibody response in vaccinated animals was assessed as described previously [23]. Briefly serum samples were initially incubated at 56°C for 30 minutes to inactivate complement, followed by serial dilution (25 µl/well) with DMEM medium in 96-well “U” bottom plates. In each well an equal volume of HCMV Ad169 was added and incubated at 37°C for 2 h. This virus was then transferred to infect monolayer of human fibroblast MRC-5 cell culture in 96 well flat bottom plates with 80–90% confluence. After 2 h, plates were washed with DMEM and 200 µl DMEM with 10% FCS were added to each well and then incubated at 37°C for 16–18 h. After incubation, cells were fixed in 100% methanol, incubated with peroxidase blocking reagent (Chemicon, S2001) and then reacted with mouse anti-CMV IE-1/IE-2 monoclonal antibody (Clone MAB810, Chemicon) followed by HRP-conjugated sheep anti-mouse Ig (Chemicon, AP326P). Finally cells were stained with DAB+ substrate (Chemicon, K3467) according to manufacturer's protocol. The numbers of nuclei with brown colour staining were counted using inverted microscope. The neutralizing titre was calculated as the reciprocal of sera dilution that gave 50% inhibition of IE-1/IE-2-expressing nuclei.Vaccinia virus recombinantRecombinant vaccinia constructs encoding HCMV antigens IE-1 (Vacc.IE-1), gB (Vacc.gB) and a negative control vaccinia virus construct made by insertion of the pSC11 vector alone, which is negative for thymidine kinase (Vacc.TK−), have been previously described [13].Protection assayHHD-2 mice were intramuscularly immunised with the indicated vaccine on day 0, followed by intraperitoneal challenge with recombinant vaccinia virus expressing different proteins from HCMV antigens (Vacc.IE1 or Vacc.gB) at a dose of 107 pfu/mouse on day 21. Mice were then sacrificed 4 days later, spleens collected to evaluate epitope-specific T cell response by IFN-γ ICS assay, ovaries collected to determine vaccinia virus load by plaque assay on monkey fibroblast CV-1 cells, and sera collected to evaluate anti-gB Ab titres by ELISA. 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+    "id": "PMC2533119",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2533119\nAUTHORS: Marie-Eve Val, Sean P. Kennedy, Meriem El Karoui, Laetitia Bonné, Fabien Chevalier, François-Xavier Barre\n\nABSTRACT:\nUnlike most bacteria, Vibrio cholerae harbors two distinct, nonhomologous circular chromosomes (chromosome I and II). Many features of chromosome II are plasmid-like, which raised questions concerning its chromosomal nature. Plasmid replication and segregation are generally not coordinated with the bacterial cell cycle, further calling into question the mechanisms ensuring the synchronous management of chromosome I and II. Maintenance of circular replicons requires the resolution of dimers created by homologous recombination events. In Escherichia coli, chromosome dimers are resolved by the addition of a crossover at a specific site, dif, by two tyrosine recombinases, XerC and XerD. The process is coordinated with cell division through the activity of a DNA translocase, FtsK. Many E. coli plasmids also use XerCD for dimer resolution. However, the process is FtsK-independent. The two chromosomes of the V. cholerae N16961 strain carry divergent dimer resolution sites, dif1 and dif2. Here, we show that V. cholerae FtsK controls the addition of a crossover at dif1 and dif2 by a common pair of Xer recombinases. In addition, we show that specific DNA motifs dictate its orientation of translocation, the distribution of these motifs on chromosome I and chromosome II supporting the idea that FtsK translocation serves to bring together the resolution sites carried by a dimer at the time of cell division. Taken together, these results suggest that the same FtsK-dependent mechanism coordinates dimer resolution with cell division for each of the two V. cholerae chromosomes. Chromosome II dimer resolution thus stands as a bona fide chromosomal process.\n\nBODY:\nIntroduction\nVibrio cholerae, the causative agent of cholera, harbors two non-homologous circular chromosomes [1]. The majority of genes believed to be necessary for the basic life processes of V. cholerae are carried on the 2.96 Mbp chromosome I, whereas the 1.07 Mbp chromosome II only harbors a few essential genes [1]. The preferential transcription of genes from chromosome II during colon colonization [2] suggests that this genomic organization is important for pathogenicity. Likewise, other bacteria with multiple chromosomes can adopt several different life cycles [3], which led to the idea that multipartite genomes offer a selective advantage for the adaptation to very different environmental conditions.Nevertheless, most bacteria harbor a single chromosome. In contrast, there is no apparent limit to the size and numbers of chromosomes harbored by eukaryotic cells. An important difference between bacteria and eukaryotes is that specific machineries appear to exist for the coordinated maintenance of each chromosome of a given bacterium, whereas eukaryotic cells possess a single global system for all chromosomes [4]–[12]. For instance, the two V. cholerae chromosomes harbor different partition systems [4],[7] and initiation of their replication is governed by different mechanisms [6],[8],[9]. In addition, many features of V. cholerae chromosome II, such as its partition system, are plasmid-like, which raised questions concerning its chromosomal nature [1],[10],[13]. Plasmid replication and segregation are generally not coordinated with the bacterial cell cycle [14], further raising questions on the mechanisms ensuring the synchronous management of chromosome I and II.A second major difference between bacteria and eukaryotes is intrinsic to the structure of chromosomes: in bacteria, chromosomes are generally covalently closed circular DNA molecules while they are linear in eukaryotes. DNA circularity can result in the formation of chromosome dimers by homologous recombination [15], which poses a barrier to the segregation of genetic information if they are not resolved before cell division (Figure 1A). Indeed, inactivation of chromosome dimer resolution (CDR) in Escherichia coli results in ∼15% cell death per generation under laboratory growth conditions [16], which corresponds to the estimated rate of chromosome dimers formed at each cell generation [17]. This prompted us to study how dimer resolution is achieved on each of the two V. cholerae chromosomes.10.1371/journal.pgen.1000201.g001Figure 1FtsK-dependent and FtsK-independent Xer recombination.A. Chromosome dimer formation and resolution in E. coli. The two homologous chromosomes are depicted by thick and thin lines, to allow for the visualization of crossovers. B. Link between the central region of XerCD-target sites (right) and the recombination pathway adopted at these sites (left). The XerCD-dif recombination complex is viewed from the C-terminal side of the recombinases, to show the C-terminal interactions of XerC and XerD. Strands cleaved by XerC and XerD in E. coli are shown with thick and thin lines, respectively. Positions of strand cleavages by E. coli XerC and XerD are indicated by white and black triangles, respectively. The WebLogo was generated using the alignment of putative dif sites from the larger chromosome of 27 γ-Proteobacteria (Text S1). The XerC-binding site, XerD-binding site and central region of dif\nEc are indicated below the alignment.The mechanism of CDR was originally elucidated in E. coli. In this organism, it depends on the addition of a crossover at dif, a 28bp site located at the opposite of the origin of replication on the chromosome, by two related tyrosine recombinases, XerC and XerD (Figure 1A; see [18] for a review). In addition, CDR depends on two activities of a cell division protein, FtsK. First, FtsK functions as a DNA pump anchored in the septum [19],[20]. It loads on DNA trapped within the division septum due to dimer formation (Figure 1A). FtsK loading is oriented by specific DNA motifs, the KOPS, which dictates the orientation of translocation (Figure 1A; [21]). KOPS are skewed on the two replichores of the chromosome with dif located at the junction of their polarity [22],[23]. Thus, dif sites carried by a dimer are brought together by FtsK translocation (Figure 1A). Second, FtsK serves to activate recombination at dif via a direct interaction with XerD [24],[25]. dif contains two 11bp binding sites for XerC and XerD, separated by a central region at the outer boundary of which recombination occurs. The interaction between XerD and FtsK allows XerD to perform a first pair of strand exchanges [20], resulting in the formation of a Holliday junction (HJ). This HJ is converted to a crossover by a second pair of strand exchanges, which is catalyzed by XerC independently of FtsK (Figure 1B, chromosomal pathway). Thus, in E. coli, the requirement for FtsK to bring dif sites together and to activate the catalytic activity of XerD permits coordination of CDR with the last stage of cell division [26].The E. coli pathway of CDR is not universal. For instance, Streptococci and Lactococci possess only a single tyrosine recombinase, XerS, for CDR [27]. Plasmid and viruses have also adopted different site-specific recombination systems to avoid multimerization of their genome. In E. coli, some of them depend on their own recombinases, such as phage P1, which encodes the Cre tyrosine recombinase [28], while others use the two Xer recombinases of their host [29],[30]. In the later case, XerC-catalysis initiates recombination independently of FtsK (Figure 1B, plasmid pathway, [18]). In this case, however, recombination requires ∼200bp of accessory sequences flanking the plasmid sites and which are bound by accessory proteins.Orthologues of E. coli xerC, xerD and ftsK are readily identified on the larger chromosome of the V. cholerae strain N16961 (xerC\nVc, xerD\nVc, ftsK\nVc, Figure S1) whereas its second chromosome does not encode any site-specific recombination system that could be implicated in CDR apart from the superintegron integrase (IntIA, Figure S1). N16961 chromosome I and II both carry dif-like sequences, dif1 and dif2, which were originally identified as integration sites for the Cholera Toxin phage, CTXφ [31]. The weak filamentous phenotype of V. cholerae cells deleted for xerC or dif1 fits with a defect in CDR [31]. However, two features of the V. cholerae Xer recombination system, which could be linked to the co-existence of distinct, non-homologous chromosomes inside the same bacterium, were intriguing. First, dif2 differs from the dif consensus of γ-Proteobacteria by 5 bases, four of which belong to the central region (Figure 1B). Such a divergence is only found on plasmid sites, which, coupled with the other plasmid-like features of chromosome II, suggested that chromosome II dimer resolution might follow a plasmid pathway. Second, it was reported that the position of cleavage of XerDVc on dif1 might differ from the one of its E. coli orthologue on dif\n[32], even if dif1 differs from the dif consensus of γ-Proteobacteria by only 2 bases (Figure 1B), further raising questions on the exact mechanisms coordinating CDR of chromosome I and II with the cell cycle.Here, we present the first formal study of CDR in V. cholerae and measure the rate of chromosome dimer formation on its two chromosomes under laboratory growth conditions. We show that the cell division protein FtsKVc is required for recombination by XerCVc and XerDVc at dif1 and dif2. In addition, we show that the activity of FtsKVc is directed by specific DNA motifs, which display the same skewed distribution on the two chromosomes, dif1 and dif2 being located at the junction of their polarity. Taken together, these results suggest that the same FtsK-dependent mechanism coordinates dimer resolution on each of the two V. cholerae chromosomes with cell division. Chromosome II dimer resolution thus stands as a bona fide chromosomal process.ResultsChromosome Dimer Formation in V. cholerae\nThe growth of V. cholerae strains deficient in CDR was directly compared to the growth of their parental strain in competition experiments in rich media (Figure 2). These experiments revealed a defect of 5.8% and 3% per cell per generation for Δdif1 and Δdif2 cells, respectively, compared to their wild type counterparts. Since these growth defects were entirely suppressed in a recA background (Figure 2), they directly reflect the rates of dimer formation on chromosome I and II, fdimer\nChr1 and fdimer\nChr2 (See Material and Methods). The 8.6% growth defect of xerCVc cells, which was also suppressed in a recA background, reflects the total rate of chromosome dimer formation in V. cholerae, fdimer\nChr1+2 (Figure 2). Interestingly, fdimer\nChr1+2 equals 1−(1−fdimer\nChr1)(1−fdimer\nChr2), indicating that dimer formation on the two V. cholerae chromosomes is independent.10.1371/journal.pgen.1000201.g002Figure 2Growth competition of V. cholerae deficient in CDR strains against their parent.f: frequency of cells that the mutant strains fail to produce at each generation compared to their parent.In Vitro Cleavage by the V. cholerae recombinases on dif1 and dif2Recombinase-mediated strand cleavage can be assayed in vitro using suicide substrates that contain a nick opposite of the position of cleavage (Figure 3A). Cleavage of the continuous strand of a suicide substrate generates a double strand break that prevents re-ligation (Figure 3B). This leads to (i) the accumulation of covalent protein/DNA complexes between the attacking recombinase and the 5′-end fragment of the continuous strand and (ii) the accumulation of free 3′-end fragments of the continuous strand (Figure 3B). XerCEc and XerDEc each cleave a specific strand on dif\nEc. The strand cleaved by XerCEc is termed Top strand. The strand cleaved by XerDEc is termed Bottom strand. Following this convention, suicide substrates in which the continuous strand is expected to be cleaved by XerCVc are called Top strand suicide substrates and suicide substrates in which the continuous strand is expected to be cleaved by XerDVc are called Bottom strand suicide substrates (Figure 3A).10.1371/journal.pgen.1000201.g003Figure 3 In vitro cleavage of dif1 and dif2 by the V. cholerae recombinases.A. Putative XerCVc and XerDVc cleavage sites on dif1 and dif2 and scheme of the suicide substrates used in this study. The top and bottom strands of dif1 and dif2 are depicted as black and grey strands. Their equivalents in dif\nEc are cleaved by XerCEc and XerDEc, respectively. Grey triangles further indicate the positions equivalent to these where XerCEc and XerDEc cleave dif\nEc. A white triangle indicates the XerDVc-cleavage position reported for dif1 [32]. Top and bottom strand suicide substrates contain a nick opposite the position expected to be cleaved by XerCVc and XerDVc if the E. coli paradigm is followed, respectively. T1, B1, T2, B2: suicide substrates on dif1 and dif2, respectively. B. Scheme of a XerC-suicide cleavage reaction. C. Covalent complex formation by MBPXerCVc and XerDVc on suicide substrates. Schemes of substrates and products are shown on the top and on the right of the gel, respectively. Suicide substrates were labeled on the 5′ side of the continuous strand, as indicated (5′*). D. Cleavage sites of XerCVc and XerDVc on dif1 and dif2. Schemes of substrates are shown on the top of the gels. Suicide substrates were labeled on the 3′ side of the continuous strand, as indicated (3′*). PNK: phosphorylation with T4 polynucleotide kinase; G+A: chemical cleavage ladder. Sequences resulting from the chemical cleavage are indicated beside the gels. Bases of the central region and of the XerCD-binding sites are indicated in black and grey, respectively. The deduced cleavage points are indicated by black triangles.Labeling the 5′-end of the continuous strand of suicide substrates allows the detection of covalent recombinase/DNA complexes (Figure 3C). The molecular weight of XerCVc and XerDVc being very similar, we used a maltose binding protein fusion of XerCVc (MBPXerCVc) in conjunction with XerDVc to avoid any confusion between the two possible covalent complexes. For both dif1 and dif2, MBPXerCVc-DNA covalent complexes accumulated when Top strand suicide substrates were used (Figure 3C, T1 and T2, respectively), indicating that XerCVc cleaves the Top strands of dif1 and dif2. Furthermore, XerDVc-DNA covalent complexes accumulated when Bottom strand suicide substrates were used (Figure 3C, B1 and B2), indicating that XerD cleaves the bottom strands of dif1 and dif2.The position of cleavage of XerCVc and XerDVc were then determined by comparing of the length of the free DNA fragments liberated by recombinase cleavage to a ladder obtained by chemical cleavage at purine bases of the suicide substrates (Figure 3D). To this aim, the continuous strands of the suicide substrates were labeled on their 3′ end. Cleavage by tyrosine recombinases generates a 5′OH DNA extremity whereas chemical cleavage leaves a 5′ phosphate. As a consequence, the free DNA fragments had to be first phosphorylated by kinase treatment (Figure 3D, PNK) in order to be compared with the chemical cleavage ladder (Figure 3D, G+A). We thus found that XerCVc and XerDVc cleave DNA at the junction between their respective binding site and the central region of dif1 and dif2 (Figure 3D, black arrows).FtsK-Dependent Recombination at dif1 and dif2Analysis of the DNA sequence immediately upstream and downstream of dif1 and dif2 in different Vibrio species did not reveal any conserved motifs that could serve to bind accessory proteins (data not shown). FtsKVc was thus left as the most likely candidate for activation of Xer recombination at both sites. To test this possibility, we reconstituted the V. cholerae Xer system in E. coli cells deleted for their natural FtsK/XerCD system. We used a xerC and xerD E. coli strain, which was also ftsKC\n−. This strain produces only the N-terminal domain of FtsKEc, essential for viability [33], but lacks production of the C-terminal domain of FtsKEc, which is necessary for recombination at dif\nEc\n[34]. XerCVc was expressed in conjunction with XerDVc from the chromosomal E. coli xerC promoter. The production of FtsKVc was controlled by placing the full length ftsKVc ORF under an arabinose-inducible promoter on a high-copy number plasmid. A low-copy plasmid carrying two recombination sites in direct repeats was used as a reporter. Recombination between the two repeated sites results in the excision of the intervening DNA, which can be monitored by agarose gel electrophoresis. For both dif1 and dif2, the amount of recombination correlated with the amount of arabinose used for induction, indicating that Xer recombination at dif1 and dif2 depends on FtsKVc (Figure 4A).10.1371/journal.pgen.1000201.g004Figure 4FtsKVc-dependent recombination at dif1 and dif2.A. Reconstitution of V. cholerae Xer recombination at plasmid-borne dif1 and dif2 sites in E. coli cells. Top panel: gel showing a typical result. A scheme of the substrate and product bands is shown beside the gel. dif sites are represented by triangles. Bottom panel: quantification plot displaying the mean and standard deviations of at least three independent experiments. B. Recombination by wild-type (+) and catalytically inactive (YF) recombinases. HJ: HJ intermediate.To determine the order of the strand exchanges in the recombination reactions, we monitored plasmid recombination in a set of four strains encoding either wild-type XerCVc and XerDVc or the XerCYF\nVc and XerDYF\nVc mutants, in which the catalytic tyrosine is replaced by a phenylalanine (Figure 4B). For both dif1 and dif2, no resolution product or HJ intermediate were detected in XerDYF\nVc cells (Figure 4B, lane 2, 4, 6 and 8). In contrast, we could detect the accumulation of a HJ intermediate in XerCYF\nVc XerDVc cells (Figure 4B, lane 3 and 7), indicating that XerDVc mediates the first pair of strand exchanges during both dif1 and dif2-recombination. Recombination products were likely still observed in XerCYF\nVc XerDVc cells since other cellular processes than Xer recombination are capable of resolving HJs [18]. However, the amount of product was considerably decreased, indicating that intermediate HJs are preferentially resolved to crossovers by the action of XerCVc.All together, these results indicate that FtsKVc activates recombination at dif1 and dif2 by promoting the exchange of a first pair of strands by XerDVc.Species-Specificity in Xer Recombination ActivationSeveral residues implicated in the interaction between E. coli XerD and FtsK have been mapped [24],[25]. These residues are not entirely conserved between the V. cholerae and E. coli proteins (Figure 5A), suggesting that the interactions between the translocase and the recombinases might be specific in these two species. Nevertheless, both FtsKEc and FtsKVc could activate recombination by XerCDEc and XerCDVc at dif\nEc, dif1 and dif2 (Figure 5B). However, the efficiency of recombination varied for each site and for each pairing of translocase/recombinases. XerCDEc-recombination at dif\nEc and dif1 reached 80% of efficiency whether FtsKEc or FtsKVc were produced (Figure 5B, XerCDEc, dif1 and dif\nEc). In contrast, XerCDEc-recombination at dif2 was more efficient when activated by FtsKEc than FtsKVc (Figure 5B, XerCDEc, dif2). In addition, it did not reach 80% efficiency, even in the presence of the cognate partner translocase, FtsKEc. XerCDVc-recombination at dif\nEc, dif1 and dif2 reached 80% of efficiency (Figure 5B, XerCDVc). However, this required the presence of FtsKVc. XerCDVc-recombination at dif2 even fell below 20% when activated by FtsKEc. Thus, the effect of species-specificity is more pronounced on dif2 than on dif1.10.1371/journal.pgen.1000201.g005Figure 5Species specificity in Xer recombination.A. Amino acid residue conservation in the γ-domain of FtsK and in the C-terminal tail of XerD. Numbers indicate the position of the first and of the last residues of the alignments in the amino acid sequence of the V. cholerae proteins. Positions of full conservation and of strong or weaker groups of conservation are indicated by stars, semi-colons or dots, respectively, following the Clustal 1.83 scheme. Black bars and vertical arrows indicate residues implicated in FtsK-XerD interaction and in KOPS recognition in E. coli, respectively. B. Species-specificity in Xer recombination on plasmid-borne dif1, dif2 and dif\nEc sites. The mean and standard deviation of at least three independent experiments are plotted. ND: not determined. C. FtsK-independent recombination at dif\nEc by wild-type (+) and catalytically inactive (YF) V. cholerae recombinases. D. FtsK-independent recombination by the V. cholerae recombinases on hybrid dif sites.Importance of the Sequence of the Resolution Sites for the Stringent Control of Xer RecombinationWe noticed that the V. cholerae recombinases could promote recombination between dif1 sites in the absence of FtsK production, albeit to a very low level (Figure 5B, XerCDVc, dif1, No FtsK). This was further exemplified on dif\nEc substrates, in which 53% of recombination was observed without FtsK expression (Figure 5B, XerCDVc, dif\nEc, No FtsK). Resolution products were detected in the absence of XerC catalysis (Figure 5C, XerCYF\nVc strains) but not in the absence of XerD catalysis (Figure 5C, XerDYF\nVc strains), signifying that XerDVc catalysis initiated recombination. dif1 differs from the γ-Proteobacteria consensus by only 2 bp, the substitution of A17 by G and the substitution of A10 by T (Figure 5D). We therefore analyzed FtsK-independent XerCDVc recombination at hybrid sites between dif, dif1 and dif2 to identify residues important for the above observation (Figure 5D). A site carrying the single [G-A]17 substitution promoted a much higher level of FtsK-independent recombination (dif12), while recombination at sites carrying the [T-A]10 and [G-A]1 substitutions was not altered (dif13 and dif14). However, the cumulative substitutions of [G-A]17 and [T-A]10 increased FtsK-independent recombination to a level equivalent to dif\nEc-recombination (dif15). In addition, when T10 was altered to A in dif2, we observed a faint recombination product (dif23), which was significant since FtsK-independent recombination was never observed at dif2. Thus, G17 in the central region of dif1 and T10 in the XerC-binding site of dif1 and dif2 appear to have an important role in maintaining Xer recombination under the tight control of FtsK in V. cholerae.\nV. cholerae FtsK Orienting Polar SequencesWe next investigated if FtsKVc could serve to bring together the CDR sites carried by dimers of chromosome I or by dimers of chromosome II. Several key residues implicated in KOPS recognition have been identified in the γ domain of FtsKEc (Figure 5A; N1296; R1300; E1303; [35]). The conservation of these residues in FtsKVc suggested that it could recognize the same motifs (Figure 5A; N926; R930; E933). If this was indeed the case, replacing the C-terminal domain of FtsKEc with the one of FtsKVc should completely rescue CDR in E. coli cells since FtsKVc fully activates recombination by XerCDEc at dif\nEc (Figure 5B, XerCDEc, dif\nEc, FtsKVc). Indeed, the fitness of such cells equaled the fitness of wild-type E. coli cells in growth competition experiments (Figure 6A, NLCVc and NLCEc), in contrast to cells only expressing the N-terminal domain of FtsKEc or a fusion with the C-terminal domain of H. influenzae FtsK (Figure 6A, N and NLCHi).10.1371/journal.pgen.1000201.g006Figure 6\n V. cholerae FtsK Orienting Polar Sequences.A. Growth competition of E. coli cells encoding FtsK hybrids. N: cells carrying a complete deletion of the C-terminal domain and linker region of FtsKEc; NLCEc: cells carrying full length FtsKEc; NLCVc and NLCHi: cells in which the C-terminal domain FtsKEc was replaced by the one of FtsKVc and FtsKHi, respectively. f: frequency of cells that the parental N strain fails to produce at each generation compared to the FtsK hybrids. B. 5′-GGGCAGGG-3′ inhibits recombination activation by FtsKVc. Plasmid recombination at E. coli dif by XerCDEc was induced with 0.5% arabinose. Ec[NRE]: FtsK50C\nEc[NRE]; Vc[NRE]: FtsKVc[NRE]. KOPS-0: substrate without GGGCAGGG sequences; KOPS-2: substrate with triple overlapping GGGCAGGG sequences in the non-permissive orientation on both sides of the two dif sites. C. Scheme of the two V. cholerae chromosomes showing the distributions of the GGGCAGGG and GGGNAGGG motifs. Upper bars: motifs found in the leading strand; Lower bars: motifs found in the lagging strand. Number, frequency, skew and skew significance (p-skew) are indicated for each motif. Recently acquired genomic regions are indicated (superintegron, CTX and TLC prophages and the Vibrio Pathogenicity Island VPI).To test for the ability of FtsKVc to specifically recognize one of the E. coli KOPS motifs, we compared the efficiency with which it activates E. coli Xer recombination between plasmid-borne difEc sites flanked or not by the 5′-GGGCAGGG-3′ motif in an orientation that should prevent it from translocating towards difEc (Figure 6B, KOPS-2 and KOPS-0, respectively). Here we observed that the efficiency of recombination dropped significantly on KOPS-2 when FtsKEc or FtsKVc were used as activators (Figure 6B, FtsKEc and FtsKVc). We then engineered an allele of ftsKVc carrying identical mutations to the one shown to abrogate KOPS recognition in FtsKEc\n[35]. No difference in recombination efficiency was noticeable between KOPS-0 and KOPS-2 when using this allele or its E. coli homologue (Figure 6B; FtsK50C\nEc[N1296A; R1300A; E1303A] and FtsKVc[N926A; R930A; E933A]). We conclude that FtsKVc directly recognizes the GGGCAGGG motif and that recognition engages amino acids N926, R930 and E933.We decided therefore to analyze the skew and frequency of the GGGCAGGG motif on chromosome I and II. GGGCAGGG is highly polarized on both chromosomes with statistically significant skews (Figure 6C). On chromosome I, the skew switches precisely at dif1 whereas on chromosome II one motif is present on the reverse orientation a few kb before dif2 (Figure 6C). However, it has been shown in E. coli that a single non-permissive KOPS motif in the vicinity of dif is not sufficient to impair recombination [23]. The frequency of GGGCAGGG is low on both V. cholerae chromosomes (Figure 6C), suggesting that this motif is not sufficient by itself to provide polar orientation of FtsKVc. We therefore analyzed the distribution of all octamers motif families with one degenerated position on both chromosomes. We ranked potential candidates according to their skew significance keeping only families that had a skew of at least 80% and a frequency of at least once every 30 kb. Only one family (GGGNAGGG) was among the 10 best candidates of both chromosomes. This family is highly skewed, frequent (Figure 6C) and contains the experimentally active GGGCAGGG motif. Taken together, these results suggest that the GGGNAGGG motifs might function as KOPS in V. cholerae.DiscussionA Common Cell Division-Coordination Mechanism for Dimer Resolution of the Two V. cholerae ChromosomesThe strand exchanges catalyzed by XerCVc and XerDVc occur at the junction between their respective binding site and the central region of dif1 and dif2, as previously reported for the E. coli recombinases on dif (Figure 3). FtsKVc promotes recombination at both sites by activating a first pair of strand exchanges mediated by XerDVc (Figure 4), thanks to a species-specific interaction with the recombinases (Figure 5). In addition, GGGNAGGG motifs seem to function as FtsKVc-Orienting Polar Sequences, their frequency and distribution on the two V. cholerae chromosomes suggesting that the FtsKVc-translocase activity helps bring CDR sites together when dimers are formed on chromosome I or on chromosome II (Figure 6). We conclude that the same FtsK-dependent mechanism controls dimer resolution on each of the two V. cholerae chromosomes. We have previously shown in E. coli that the requirement for FtsK to activate Xer recombination delays CDR to the time of septum closure [26], which is likely to also hold true in V. cholerae. Thus, the study of CDR provides the first example of a cell cycle coordination mechanism shared by the two V. cholerae chromosomes, which is similar to the way chromosomal maintenance processes are coordinated within the cell cycle of eukaryotes.Dimer Formation Is Linked to Replicon SizeMany bacteria harbor multiple chromosomes, which seems an important determinant of their individual life styles. A few bacterial species harbor linear replicons in addition to circular, such as Agrobacterium tumefaciens and the Borrelia species [3]. In the vast majority of cases, however, the multiple chromosomes harbored within a bacterium are circular. Maintenance of circular replicons requires the resolution of dimers created by homologous recombination events. In V. cholerae, 5.8% of dimers per cell per generation are formed on the 2.96 Mbp chromosome I and 3% of dimers are created on the 1.07 Mbp chromosome II (Figure 2). Under similar growth conditions, 15.6% of dimers are generated on the 4.6Mbp E. coli chromosome (Figure 6). These results suggest that dimer formation increases with replicon size, possibly reaching a theoretical upper limit of 50% for very large replicons. In addition, the rate of dimer formation seems to vary exponentially with replicon size for small replicons. Based on this hypothesis, the frequency of chromosome dimer formation in V. cholerae would be 11% per cell generation if it carried a single circular chromosome of 4.03Mbp. Instead, we measured a total rate of 8.6% for the two chromosomes (Figure 2). Thus, the particular genomic organization of the Vibrios seems to minimize chances for chromosome dimer formation, which is theoretically beneficial.Generalization to Other Bacteria with Multiple ChromosomesPutative dif sites were readily identified on each of the two chromosomes harbored by 7 additional γ-Proteobacteria (Figure 7 and Figure S2). To determine dif sites in β- and α-Proteobacteria, we generated a profile Hidden Markov Model (HMM) based on the alignment of the putative CDR sites found in the larger chromosome of 27 γ-Proteobacteria using the program HHMER. We then compared each sequence by hand to ensure the proper 6 bp spacing between the putative XerC and XerD binding sites. Putative dif sites were thus identified on each of the multiple chromosomes harbored by 10 β-Proteobacteria species and 5 α-Proteobacteria species (Figure 7 and Figure S3 and S4). A single pair of recombinases orthologous to XerC and XerD was found in each of the 22 additional γ-, β- and α-Proteobacteria harboring multiple chromosomes, suggesting that a single pair of recombinases ensures dimer resolution of each of their non-homologous chromosomes. FtsK orthologues were also found. In addition, putative dif sites fell within 10 kb of the GC-skew inflection point (data not shown), suggesting that dimer resolution is under the control of an FtsK-like homologue in all these species. Thus, the adoption of an FtsK-dependent dimer resolution system could be a key evolutionary step in the maintenance of large circular replicons.10.1371/journal.pgen.1000201.g007Figure 7The non-homologous chromosomes of Proteobacteria with multipartite genomes carry divergent dif sites.Alignment of the chromosome dimer resolution sites of a few Proteobacteria harboring a single or multiple chromosomes. Bases identical to the γ-Proteobacteria dif consensus are shaded in black. Species abbreviations follow the KEGG convention.Tuning of V. cholerae CDR to Achieve Efficient Recombination on Divergent dif SitesThe sequence of Xer target sites, and especially of their central region, is a crucial determinant in the outcome of recombination [36],[37]. Indeed, the central region of dif sites found in Proteobacteria with a single chromosome showed a high degree of conservation, most β- and γ-Proteobacteria harboring a ‘canonical’ 5′-TGTATA-3′ motif (Figure 7 and Figure S2 and S3), suggesting that there is a selective pressure on the sequence of the dif central region. This is further illustrated by the lower recombination efficiency of the E. coli system on dif2 compared to dif1 (Figure 5). In this regard, the V. cholerae Xer recombination system is remarkable since identical recombination efficiencies were obtained with the same pair of recombinases on dif1 and dif2 (Figure 4 and 5). However, XerCDVc-mediated recombination at dif2 required a tighter interaction between the recombinases and their partner translocase than at dif1, since FtsKEc promoted 50% of recombination at dif1 but less than 20% at dif2 (Figure 5B, XerCDVc, FtsKEc). In addition, a few alterations in the sequence of dif1 and dif2 decreased the stringency of the control exerted by FtsKVc (Figure 5D), highlighting the extremely fine tuning of the different components of the V. cholerae CDR system.Non-Homologous Chromosomes Carry dif Sites with Divergent Central RegionsWe observed that in Proteobacteria with multiple chromosomes, the central regions of dif sites from non-homologous chromosomes are divergent, as in V. cholerae (Figure 7 and Figure S2, S3, S4). A single exception was found in Burkholderia xenovarans, in which two of the three chromosomes of the bacterium harbor a resolution site with an identical central region. We reasoned therefore that some selective pressure imposes the divergence of the central regions of CDR sites carried by the different, non-homologous chromosomes of bacteria with multipartite genomes, which competes with the selective pressure for dif central regions to adopt the preferential 5′-TGTATA-3′ motif. Indeed, the presence of dif sites with identical central regions on two non-homologous chromosomes could lead to the formation of chromosome fusions by Xer recombination, which would disrupt the selective advantage brought by the multipartite genomic organization. In support of this hypothesis, preliminary experiments indicate that harmonization of the two V. cholerae dif sites leads to chromosomal fusions (Val and Barre, unpublished observations). We are currently investigating how these fusions are formed and the consequences of harboring identical dif sites on separate chromosomes.Materials and MethodsStrains, Plasmids, and MediaAll growth experiments were done in LB-Lennox. Strains and plasmids are listed in Text S1. Briefly, V. cholerae strains were derived from N16961 [1] by allele exchange using pDS132 derivatives [38] and E. coli β2163 as a donor strain [39]. E. coli strains used for in vivo plasmid resolution assays and for growth competition were engineered as previously described in [25],[40]. Mutations were confirmed by PCR and sequencing.Growth Competition AssayFor growth competitions, E. coli cells were grown at 37°C with a 1000× dilution in fresh media every 12h [40]. Because of their higher growth rate, V. cholerae cells were grown at 30°C with a 10000× dilution every 12h. The numbers of CFU of mutated and parental cells in the cultures were determined by plating on cognate antibiotic plates every 12 or 24h, depending on the mutant growth defect. These numbers were used to calculate the number of generation of the parent cells between each time points and the CFU ratio of mutated versus parent cells at each time point. This ratio varies exponentially with the number of generations. The proportion of cells that the mutant strain fails to produce at each doubling time of its parent is deduced from the coefficient of this exponential. This ratio is a good estimation of the rate of dimer formation (Text S1).In Vitro Xer Assays\nV. cholerae MBP-XerD and MBP-XerC recombinases were purified using nickel, amylose and heparin columns. The MBP tag was removed by thrombin digestion. dif1 and dif2 synthetic suicide substrates (Text S1) were obtained by annealing synthetic oligonucleotides purified by PAGE. 5′-end labeling of oligonucleotides was performed using T4 DNA polynucleotide kinase and [32P] γ-ATP and 3′end labeling using terminal transferase and [32P] α-ddATP. Reactions were performed in 20 mM Tris-HCl (pH 7.5), 50 mM NaCl, 0.1mM EDTA, 1 μg/ml of BSA, 40% glycerol and 0.2 pmol of radiolabeled probe for 2 hours at 37°C. Covalent complexes were analyzed by 12% SDS-PAGE and cleavage sites by 12% urea-PAGE. Radioactivity was detected on a STORM (GE Healthcare).In Vivo Plasmid Resolution Assays\nE. coli cells were transformed with the FtsK expression vector and then with the Xer recombination reporter plasmid, as described in [25]. 10 transformant colonies were pooled in 1 ml of LB, diluted 100× in LB and grown to 0.6 OD at 37°C. Cells were then grown for an extra 2 hours at 37°C in the presence of 0.5% arabinose to induce FtsK production, unless otherwise indicated. Plasmid DNA was hydrolyzed with NdeI (single cutter). Recombination efficiency was computed as the amount of replicative product over the sum of the amount of substrate and of replicative product, which were separated by agarose gel electrophoresis and detected with SybrGreen staining using a LAS-3000 (Fuji Life Science).Bioinformatics Analysis of Motifs DistributionLeading strands were defined as the DNA strand reported in Genbank files downstream of the replication origin up to the terminus and the reverse complement strand from the terminus to the origin. The terminus position was chosen as the first nucleotide of the CDR site. Skew statistical significance was assessed by calculating the probability that the observed skew occurred by chance taking into account the fact that G-rich motifs are likely to be more frequent on the leading strand because of GC skew, as previously described [41]. Analysis on chromosome II was performed on a chimeric chromosome where the superintegron has been removed because this element carries more than 100 repetitions of the attC integration site, which hides the signal provided by octamer motifs.Supporting InformationFigure S1XerCD and FtsK tree.(2.80 MB TIF)Click here for additional data file.Figure S2dif sites in gamma-Proteobacteria.(6.03 MB TIF)Click here for additional data file.Figure S3dif sites in beta-Proteobacteria.(5.50 MB TIF)Click here for additional data file.Figure S4dif sites in alpha-Proteobacteria.(3.18 MB TIF)Click here for additional data file.Text S1Supplementary methods.(0.12 MB DOC)Click here for additional data file.\n\nREFERENCES:\n1. 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DraperGCMcLennanNBeggKMastersMDonachieWD\n1998\nOnly the N-terminal domain of FtsK functions in cell division.\nJ Bacteriol\n180\n4621\n4627\n9721304\n34. BarreFXAroyoMCollomsSDHelfrichACornetF\n2000\nFtsK functions in the processing of a Holliday junction intermediate during bacterial chromosome segregation.\nGenes Dev\n14\n2976\n2988\n11114887\n35. SivanathanVAllenMDde BekkerCBakerRArciszewskaLK\n2006\nThe FtsK gamma domain directs oriented DNA translocation by interacting with KOPS.\nNat Struct Mol Biol\n13\n965\n972\n17057717\n36. RecchiaGDAroyoMWolfDBlakelyGSherrattDJ\n1999\nFtsK-dependent and -independent pathways of Xer site-specific recombination.\nEMBO J\n18\n5724\n5734\n10523315\n37. CapiauxHLesterlinCPeralsKLouarnJMCornetF\n2002\nA dual role for the FtsK protein in Escherichia coli chromosome segregation.\nEMBO Rep\n3\n532\n536\n12034757\n38. PhilippeNAlcarazJPCoursangeEGeiselmannJSchneiderD\n2004\nImprovement of pCVD442, a suicide plasmid for gene allele exchange in bacteria.\nPlasmid\n51\n246\n255\n15109831\n39. DemarreGGueroutAMMatsumoto-MashimoCRowe-MagnusDAMarlièreP\n2005\nA new family of mobilizable suicide plasmids based on the broad host range R388 plasmid (IncW) or RP4 plasmid (IncPa) conjugative machineries and their cognate E.coli host strains.\nResearch in Microbiology. In press\n40. BigotSCorreJLouarnJCornetFBarreFX\n2004\nFtsK activities in Xer recombination, DNA mobilization and cell division involve overlapping and separate domains of the protein.\nMol Microbiol\n54\n876\n886\n15522074\n41. HalpernDChiapelloHSchbathSRobinSHennequet-AntierC\n2007\nIdentification of DNA motifs implicated in maintenance of bacterial core genomes by predictive modeling.\nPLoS Genet\n3\n1614\n1621\n17941709"
+}

batch_11/PMC2533121.json

@@ -0,0 +1,4 @@
+{
+    "id": "PMC2533121",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2533121\nAUTHORS: Heather B. Jaspan, Lyen C. Huang, Mark F. Cotton, Andrew Whitelaw, Landon Myer\n\nABSTRACT:\nBackgroundSerious bacterial infections are a major source of morbidity and mortality in HIV-infected children. The spectrum of disease is wide, and responsible organisms vary according to setting. The use of antibiotic prophylaxis and the emergence of multi-drug resistant bacteria necessitate examination of responsible organisms and their antibiotic susceptibility.Methodology/Principal FindingsA retrospective cohort study of all HIV-positive pediatric admissions at an urban public sector hospital in Cape Town between January 2002 and June 2006 was conducted. Children between the ages of one month and nine years with laboratory confirmed HIV status, serious bacterial infection, and a hospital length of stay of 5 days or more, were eligible for inclusion. Organisms isolated from blood, urine, and cerebral spinal fluid cultures and their antimicrobial susceptibility were examined, and compared according to timing of isolation to distinguish nosocomial versus community-acquired. One hundred and forty-one children were identified (median age 1.2 years), 39% of whom were on antiretrovirals started before or during this hospitalization. Bacterial infections involved all organ systems, however pneumonia was most common (67%). S. pneumoniae and S. aureus were the most common gram positive and K. pneumoniae was the most common gram negative organism. K pneumoniae isolates were resistant to many first and second line antibiotics, and were all considered nosocomial. All S. aureus isolates were methicillin resistant, some of which were community-acquired.Conclusions/SignificanceBacterial infections are an important source of co-morbidity in HIV-infected children in resource-limited settings. Clinicians should have a low threshold to initiate antibiotics in children requiring hospitalization. Broad-spectrum antibiotics should be used judiciously. Clinicians caring for HIV-infected children should be cognizant of the most common organisms affecting such children, and of their local antimicrobial susceptibilities, when treating empirically for serious bacterial infections.\n\nBODY:\nIntroductionBacterial infections are a major source of morbidity and mortality in HIV- infected children, causing . a wide spectrum of diseases, many of which are included in the WHO and CDC staging systems[1], [2] In an early USA study predating both trimethoprim-sulfamethoxazole (TMP-SMX) and antiretroviral therapy (ART)[3], there were 160 episodes of minor and 48 serious bacterial infections (SBI) per 100 patient-years in HIV-infected children, demonstrating the extremely high burden of bacterial morbidity. A similar study performed after the introduction of TMP-SMX and zidovudine monotherapy documented a two-year SBI rate of 45% among children receiving TMP-SMX.[4]. In a randomized controlled trial of TMP-SMX prophylaxis for HIV-infected children in Zambia, the SBI admission rate was 19 per 100 child-years in the TMP-SMX group and 29 in the placebo group (P = 0.09), and the cumulative two-year probability of dying in hospital from SBI (predominantly pneumonia) was 7% on TMP-SMX and 12% on placebo (P = 0.08).[5] Thus, data from varied settings documented high rates of SBI in children and a protective effect of TMP-SMX.Since the widespread availability of antiretroviral therapy (ART), there has been a marked decrease in the morbidity and mortality of bacterial infections in both resource rich and resource limited settings [6], [7]. In the USA, pneumonia is still the most common bacterial infection in HIV-infected children on ART, however, in comparison with the pr-ART era, the incidence has decreased from 11 to 2 events per 100 person years.[8] In a Californian cohort, bacterial infections accounted for 60 hospitalizations for 64 children in 1994, versus only 8 for 101 children in 2001.[9] The hospitalization rate for SBI was only 14.2 per 100 person years for a Thai cohort of children on ART.[10] Nevertheless, bacterial infections remained the most common reason for hospitalization.HIV-infected children have a greater risk of bacterial infections than their HIV-negative counterparts[11], and these infections are more invasive, more likely to disseminate, and have worse outcome in HIV-infected children.[11]–[13] Also, HIV-infected children often have multiple diagnoses and polymicrobial infections.[11], [12] All organ systems can be involved by bacterial infections, and concomitant bacteraemia is common. Abscess formation in internal organs and skin can also occur. Otitis media (OM), acute sinusitis and mastoiditis are also common. By far the most frequent cause of bacterial morbidity in all HIV-infected children is pneumonia, both with and without TMP-SMX prophylaxis.[5]\nThe emergence of antimicrobial resistant organisms is a global problem, not restricted to children with HIV. However, the widespread use of TMP-SMX for prophylaxis may exacerbate antimicrobial resistance. In a South African study, only four of 26 S. pneumoniae from HIV-infected children with community acquired pneumonia were sensitive to TMP-SMX.[11] Resistance was unaffected by TMP-SMX prophylaxis, arguing that resistance may be firmly established in the community. The antimicrobial resistance patterns from blood cultures from HIV infected and uninfected children have been compared in only a few settings. The resistance of both S. pneumoniae and S. aureus to TMP-SMX was significantly higher in the HIV-infected group's isolates in Soweto [14]. In three African studies, almost 15% of the isolates from HIV-infected children were multiply resistant E. coli or Methicillin resistant staphylococcus aureus (MRSA). [14]–[16] However, in the Ugandan children, organisms from HIV-infected children were either more sensitive or had the same profile as uninfected children.[16] Therefore it is unclear how widespread antimicrobial resistance is in HIV-infected children.MethodsObjectivesWe sought to evaluate the spectrum of bacterial infection, causative organisms, and antibiotic resistance patterns of an inpatient cohort of HIV-infected children.ParticipantsWe conducted a retrospective cohort study of all HIV-positive pediatric admissions to a Pediatric ward in an urban public sector hospital in Cape Town between January 2002 and June 2006. Over 50% of all admissions are HIV-infected. The referral population is a largely black African population living in rapidly growing settlements with residents of predominantly low socioeconomic status. Data were collected from paper charts and computer records. Where more than one hospitalization per patient occurred, only the first was examined.Children between one month and nine years of age with laboratory confirmed HIV-infection, SBI and hospitalization for 5 or more days, were eligible for inclusion.Description of investigationsThe following were considered SBI: bacterial sepsis, bacterial pneumonia, urinary tract infection, and/or meningitis. For bacterial sepsis, children had organisms identified on blood culture. Diagnosis of bacterial pneumonia was made by clinical findings, laboratory results including high white blood cell count with neutrophil predominance, lobar consolidation on chest radiograph, and blood cultures when available. For meningitis, either an organism was isolated from cerebrospinal fluid or the chemistry and cell counts were suggestive of bacterial infection. Coagulase negative staphylococcus only included as a pathogen if isolated from more than one culture or from a patient with indwelling invasive lines. All S. aureus isolates were considered pathogenic, as the patients were ill. An infection was considered community acquired if symptomatic at admission or if cultures taken at or before 48 hours after admission were positive. If positive cultures or new symptoms appeared after 48 hours, the infection was considered nosocomial or hospital acquired [17].Clinical specimens submitted for bacterial culture were inoculated into appropriate media depending on the nature of the specimen. Organisms were identified by standard biochemical and/or serological methods. Antimicrobial susceptibility testing was done by the Kirby-Bauer disc diffusion technique, and MICs (if necessary) were performed using E-tests. Susceptibilities were not available for all specimens from 2002–2003. Therefore, susceptibilities represent more recent specimens.EthicsApproval for this study was obtained from the University of Cape Town Faculty of Health Sciences Research Ethics Committee. Exemption from informed consent was granted as this was a retrospective study with no identifying data.ResultsCohort DemographicsOne hundred and forty-one children met inclusion criteria. The median age was 1.2 years (IQR 0.5–2.3) and 65 (46%) were female (table 1). The median weight on admission was 6.7 kg (IQR 4.7–9.6)), 16 (11.4%) with a diagnosis of marasmus and a further 27 (19%) labeled as failure to thrive.10.1371/journal.pone.0003260.t001Table 1Demographics and spectrum of serious bacterial disease in hospitalized children with HIV.Characteristicn (%)GenderMales76 (54)Females65 (46)On ART55 (39)On TB medication39 (28)Mortality13 (9)Age at admission (yrs)1.2 (0.5–2.34)Median (IQR)Weight at admission (kg)6.7 (4.7–9.6)Median (IQR)Temp. at admission (°C)37.3 (36.5–38.3)Median (IQR)Birth weight (kg)2.8 (2.8–3.0)Mean (95% CI)Hemoglobin on admission9.1 (8.1–10.3)Median (IQR)Concomitant DiagnosesTB40 (28)TB meningitis2 (1)Gastroenteritis54 (38)PEM16 (11)Fungal sepsis4 (2.3)Failure to thrive29 (21)Bacterial Infections40 (28)Bacterial sepsis74 (52)Pneumonia95 (67) % Bacteremic pneumonia50Bacterial meningitis4 (3) % Bacteremic meningitis50Urinary tract infection15 (11) % Bacteremic UTI87Skin or soft tissue, including abscess2 (1.5)Osteomyelitis or septic arthritis1 (0.7)ART–antiretroviral therapy, TB–Tuberculosis, PEM-Protein energy malnutrition, IQR–Interquartile range.Approximately one third of children received ART(n = 55). However, the median time on ART was only 6 days (IQR -8–35), and many were started during this hospitalization. There were 40 children on tuberculosis treatment; some children were receiving both ART and TB treatment (n = 16; 11.3%). The median CD4 percentage was 17.2% (IQR 9.7–22.7), with a median absolute CD4 count of 509 cells/mm3 (IQR 244–759). The median hospital length of stay was 13 days (IQR 6–34 days). There were 13 deaths in hospital with the remainder being discharged.Spectrum of DiseaseA lower respiratory tract infection was diagnosed in 95 (67%) of the children, while 74 (53%) had bacterial sepsis. The most prevalent bacterial infections in this cohort of hospitalized children were pneumonia and sepsis (Table 1). However, most other organ systems were affected, including the central nervous system (meningitis), lung, mastoid, bone and joints. A high proportion of patients with pneumonia, meningitis, and urinary tract infection had concomitant bacteremia. Mycobacterium tuberculosis was a frequent concomitant infection. Eight deaths had dual diagnoses of sepsis and pneumonia, and one had both sepsis and a UTI.Responsible OrganismsThe relative frequencies of specific bacterial isolates from blood, urine and CSF cultures of HIV-infected children are shown in table 2 (excluding mycobacteria). The most common pathogen isolated from blood was S. pneumoniae followed by S aureus. The most common gram negative organism was K. pneumoniae. All K. pneumoniae isolates were from blood cultures taken more than two days into the admission, implying a nosocomial origin. In contrast, all but two S pneumoniae from blood culture were within two days of admission, therefore probably community acquired. The organisms most frequently present when the outcome was fatal were S aureus (n = 3, all MRSA), Coagulase negative staphylococcus (n = 3) and K pneumoniae (n = 3). Five of the 13 children who died grew multiple pathogenic organisms in blood or urine.10.1371/journal.pone.0003260.t002Table 2Bacterial isolates from hospitalized HIV-infected children with serious bacterial infections*Blood n = 128 (%1)Urine n = 21 (%2)CSF n = 36 (%2)N (%) assumed nosocomial (>48 hours)3\n\nS. pneumoniae\n21 (16)1 (2.8)2/22 (9%)\nS. aureus\n14 (11)9/14 (64%)Coagulase negative staphylococcus4\n4 (3)4/4 (100%)\nE. coli\n5 (4)5 (24)7/10 (70%)\nK. pneumoniae\n11 (9)2 (9.5)13/13 (100%)Hemophilus spps8 (7)1 (2.8)1/9 (11%)\nS. typhi\n1 (1)0Non typhoid salmonella9 (7)1 (4.8)7/10 (70%)\nS. milleri\n1 (1)1 (100%)\nEnterococcus faecalis\n4 (3)2 (9.5)5/6 (83%)\nEnterococcus faecium\n1 (1)1 (100%)\nEnterobacter species\n6 (4)4/6 (67%)\nP. aeruginosa\n1 (1)1 (4.8)0Acinetobacter4 (3)3/4 (75%)1Percent of positive cultures, including fungal and mixed or skin flora2Percent of all cultures sent. Urine cultures were not done if urine dipstick was normal unless suspicion was high.3The proportion of nosocomial infections presented here reflects that from children hospitalized for five days or longer, and not necessarily the proportion of nosocomial infections from hospitalizations of all HIV-infected children.4Coagulase negative staphylococcus only included as a pathogen if isolated from more than one culture or with indwelling invasive lines.Susceptibility PatternsThe antimicrobial susceptibility patterns of each of the more prevalent organisms are shown in table 3. S. pneumoniae and H. influenza isolates were generally highly susceptible, whereas the majority of the K. pneumoniae isolates, most likely nosocomial, were highly resistant. All K. pneumoniae isolates were extended spectrum beta-lactamase producing; the only single antibiotic to which all K. pneumoniae isolates were susceptible was Meropenem. S. aureus whether community-acquired or nosocomial, was often resistant to TMP-SMX, and clindamycin, both frequently used antibiotics for resistant staphylococci. . Although community-acquired non-typhoid salmonella (NTS) were susceptible to first-line antibiotics such as ampicillin, the more frequently hospital-acquired organisms were more resistant. MRSA was isolated from a third of the blood cultures of children who died, as was K. pneumoniae, therefore resistant organisms were possibly responsible for a relatively high proportion of the mortality, and appropriate empiric therapy was not used.10.1371/journal.pone.0003260.t003Table 3Antibiotic susceptibilities of the most common organisms isolated from blood cultures of children with serious bacterial infections, according to time of isolation.% susceptible\nS. pneumoniae\n\nS. aureus\n\nH. influenzae\n\nE. coli\nNon-typhoid salmonella\nK. pneumoniae\n\nTime ( Total n)\n≤48 hrs (14)\n>48 hrs (2)\n≤48 hrs (3)\n>48 hrs (9)\n≤48 hrs (3)\n>48 hrs (2)\n≤48 hrs (1)\n>48 hrs (4)\n≤48 hrs (3)\n>48 hrs (6)\n≤48 hrs (0)\n>48 hrs (10)Penicillin/Ampicillin79a\n50a\n0010010005010017NA0Amoxicillin +clavulanateNTNTNTNT10010010050100100NA20b\nTMP-SMXNTNT0067007510017NA0Cloxacillin/MethicillinNTNT00NTNTNTNTNTNTNTNT3rd generation Cephalosporin93c\n100NTNT1001001005010020NA0Erythromycin865000NTNTNTNTNTNTNTNTGentamicinNTNT00NTNT10050NTNTNA10ClindamycinNTNT6733NTNTNTNTNTNTNTNTOfloxacin/CiprofloxacinNTNT336710010010075100100NA40AmikacinNTNT10078NTNT100100NTNTNA90a\nPipericillin/TazobactamNTNTNTNTNTNT10075a,c\n100100NA50a\nMeropenemNTNTNTNTNTNT100100100100NA100VancomycinNTNT100100NTNTNTNTNTNTNTNTaThe remainder were intermediate sensitivitybFive isolates intermediate susceptibilitycOne unknownNT–not testedNA–not applicableTMP-SMX–Trimethoprim sulfamethoxazoleDiscussionIn this cohort of hospitalized HIV-infected children, pneumonia was the most common SBI resulting in hospitalization. There was frequent coincident bacteremia. This is consistent with other HIV-infected pediatric cohorts described from both developing and developed settings[5], [8]. The relative prevalence of most bacterial pathogens is similar to that of HIV-uninfected children. In a large Kenyan study S. pneumoniae, H. influenzae, NTS, and E. coli were more common in HIV-infected children [18]. These organisms were commonly seen in malnourished children in the pre-HIV era. S. pneumoniae is the most common organism in other cohorts, with S. aureus, Salmonella species, and Enterobacteriaceae occurring frequently [4], [11], [14], [15]. P. aeruginosa is also seen occasionally. The proportion of H influenzae is influenced by the introduction of the H. influenzae type B (Hib) vaccine, in 1999 in South Africa.Bacteria that were assumed to be predominantly community acquired were generally susceptible to first line or narrow spectrum antibiotics (those that target generally only one class of bacteria). This included the S. pneumoniae isolates. In other settings, multi-drug resistant S. pneumoniae were increasing in frequency before introduction of the pneumococcal conjugate vaccine [19], [20]. In children with community acquired pneumonias in Soweto, South Africa, 50% of the S. pneumoniae isolates from the HIV-infected children were penicillin resistant versus 23% of the HIV-negative children's isolates.[14] The community-acquired S. aureus isolates were, however, all MRSA. The empiric treatment of community acquired bacterial infections in HIV-infected children in resource limited settings should bear in mind the antimicrobial susceptibility patterns of the region. Penicillin and ampicillin are effective against most penicillin-non-susceptible pneumococcal isolates in the case of isolated respiratory tract infections. The clinician must consider viral, fungal, and mycobacterial infections in the differential diagnosis of bacterial infections and treat accordingly.The high proportion of K. pneumoniae resistant to third generation cephalosporin is highly concerning, particularly since all these infections were acquired after two days in hospital, and therefore probably hospital-acquired. Highly resistant organisms have been described from a similar setting [15], and is likely due to extended spectrum beta lactamase production. Since not all K. pneumoniae isolates were susceptible to piperacillin-tazobactam or amikacin, clinicians should consider empiric carbapenems for suspected hospital-acquired sepsis.Nosocomial infections are more common in HIV-infected than in uninfected adults, with the most frequently isolated bacterial pathogens being S. aureus and P. aeruginosa, a high proportion of which are methicillin resistant [21]. Nosocomial infections in HIV-infected adults have a high mortality. There are few data on nosocomial bacterial infections in HIV-infected children. In a cohort of children hospitalized with TB, nosocomial infections were more common in HIV-infected patients, and more frequently fatal [22]. Although our data do not reflect the overall prevalence of nosocomial infections in hospitalized HIV-infected children due to our study design, the data may represent common resistance patterns of these organisms.The most effective public health approach to improving infectious disease burden is vaccination. Effective vaccines are licensed for Hib and S. pneumoniae. Both are polysaccharide protein conjugate vaccines. The Hib vaccine is now widely available including in developing countries. Although the estimated efficacy of the Hib vaccine is decreased and the risk for vaccine failure increased in HIV-infected children,[23], [24] the introduction of Hib vaccine into countries with high HIV prevalence has greatly decreased Hib disease burden.[25] In 2000, a pneumococcal conjugate vaccine (PCV) was licensed in the USA. Although PCV is less immunogenic in HIV-infected children than non-infected children,[26] this also seems to improve in children receiving ART.[27] A similar 9-valent PCV has been extensively studied in South Africa, and similar efficacy is seen in these HIV-infected infants.[28] Unfortunately, PCV is not widely accessible to children in less resourced countries, and its access should become a global priority.These data confirm that pneumonia is the most common SBI in HIV-infected children, but with a wide spectrum of presentation. The predominance of MRSA is alarming. The high proportion of nosocomially acquired highly resistant K. pneumoniae, suggest that infection control practices should be implemented to avoid the spread of antibiotic resistance. While overuse of broad-spectrum antibiotics should be avoided, judicious limited use is indicated for suspected nosocomial sepsis.LimitationsThe limitations of these data are that they are retrospective and therefore incomplete. In addition, the isolation of organisms is dependant on adequate blood volumes and rapid transport to the microbiology laboratory, both of which may have been limited in the setting of sick, malnourished children in a resource-limited hospital. Our findings may not be generalizable to other better resourced settings such as those with more nursing staff, and less ward crowding, or where all HIV-infected children are on ART.\n\nREFERENCES:\n1. CDC\n1987\nClassification system for human immunodeficiency virus (HIV) infection in children under 13 years of age.\nMMWR\n36\n225\n230\n3031443\n2. World Health Organisation\n2006\nAntiretroviral therapy of HIV infection in infants and children in resource-limited settings: towards universal access\n3. MofensonLNugentRRigauperezJ\n1991\nSerious Bacterial-Infections in Hiv-Infected Children in A Clinical-Trial of Intravenous Immunoglobulin.\nPediatric Research\n29\nA179\n4. SpectorSAGelberRDMcgrathNWaraDBarzilaiA\n1994\nControlled Trial of Intravenous Immune Globulin for the Prevention of Serious Bacterial-Infections in Children Receiving Zidovudine for Advanced Human-Immunodeficiency-Virus Infection.\nNew England Journal of Medicine\n331\n1181\n1187\n7935655\n5. MulengaVFordDWalkerASMwenyaDMwansaJ\n2007\nEffect of cotrimoxazole on causes of death, hospital admissions and antibiotic use in HIV-infected children.\nAids\n21\n77\n84\n17148971\n6. NesheimSRKapogiannisBGSoeMMSullivanKMAbramsE\n2007\nTrends in opportunistic infections in the pre- and post-highly active antiretroviral therapy eras among HIV-infected children in the Perinatal AIDS Collaborative Transmission Study, 1986-2004.\nPediatrics\n120\n100\n109\n17606567\n7. KapogiannisBGSoeMMNesheimSRSullivanKMAbramsE\n2008\nTrends in bacteremia in the pre- and post-highly active antiretroviral therapy era among HIV-infected children in the US Perinatal AIDS Collaborative Transmission Study (1986-2004).\nPediatrics\n121\ne1229\ne1239\n18450865\n8. GonaPVan DykeRBWilliamsPLDanknerWMChernoffMC\n2006\nIncidence of opportunistic and other infections in HIV-infected children in the HAART era.\nJama-Journal of the American Medical Association\n296\n292\n300\n9. VianiRMAranetaMRGDevilleJGSpectorSA\n2004\nDecrease in hospitalization and mortality rates among children with perinatally acquired HIV type 1 infection receiving highly active antiretroviral therapy.\nClinical Infectious Diseases\n39\n725\n731\n15356789\n10. PuthanakitTAurpibulLOberdorferPAkarathumNKanjananitS\n2007\nHospitalization and mortality among HIV-infected children after receiving highly active antiretroviral therapy.\nClinical Infectious Diseases\n44\n599\n604\n17243067\n11. McNallyLMJeenaPMGajeeKThulaSASturmAW\n2007\nEffect of age, polymicrobial disease,, and maternal HIV status on treatment response and cause of severe pneumonia in South African children: a prospective descriptive study.\nLancet\n369\n1440\n1451\n17467514\n12. MeyersTMPettiforJMGrayGECrewe-BrownHGalpinJS\n2000\nPediatric admissions with human immunodeficiency virus infection at a regional hospital in Soweto, South Africa.\nJournal of Tropical Pediatrics\n46\n224\n230\n10996984\n13. ZarHJHansloDTannenbaumEKleinMArgentAEleyBBurgessJMagnusKBatemanEDHusseyG\n2001\nAetiology and outcome of pneumonia in human immunodeficiency virus-infected children hospitalized in South Africa.\nActa Paediatrica\n90\n119\n125\n11236037\n14. MadhiSAPetersenKMadhiAKhoosalMKlugmanKP\n2000\nIncreased Disease Burden and Antibiotic Resistance of Bacteria Causing Severe Community-Acquired Lower Respiratory Tract Infections in Human Immunodeficiency Virus Type 1-Infected Children.\nClinical Infectious Diseases\n31\n170\n176\n10913417\n15. CottonMWassermanESmitJWhitelawAZarHJ\n2008\nHigh incidence of antimicrobial resistant organisms including extended spectrum beta-lactamase producing Enterobacteriaceae and methicillin-producing Staphylococcus aureus in nasopharyngeal and blood isolates of HIV-infected children from Cape Town, South Africa.\nPLOS Medicine\n16. BachouHTylleskarTKaddu-MulindwaDHTumwineJK\n2006\nBacteraemia among severely malnourished children infected and uninfected with the human immunodeficiency virus-1 in Kampala, Uganda.\nBmc Infectious Diseases\n6\n17. GarnerJJarvisWEmoriTHoranTHughesJJarvisW\n1988\nCDC definitions for nosocomial infections.\nAmerican Journal of Infection Control\n16\n28\n40\n18. BerkleyJLoweBMwangiIWilliamsTBauniE\n2005\nBacteremia among children Admitted to a Rural Hospital in Kenya.\nNew England Journal of Medicine\n352\n39\n47\n15635111\n19. WintersMPatrickDMarraFBuxtonJChongMIsaac-RentonJ\n2008\nEpidemiology of invasive pneumococcal disease in BC during the introduction of conjugated pneumococcal vaccine.\nCanadian Journal of Public Health\n99\n57\n61\n18435393\n20. PletzMMausUKrugNWelteTLodeH\n2008\nPneumococcal vaccines: mechanism of action, impact on epidemiology and adaption of the species.\nInternational Journal of Antimicrobial Agents March 29 Epub ahead of print\n21. FranzettiFGrassiniADegl'innocentiMBanderaAGazzolaL\n2006\nNosocomial bacterial pneumonia in HIV-infected patients: risk factors for adverse outcome and implications for rational empiric antibiotic therapy.\nInfection\n34\n9\n16\n16501896\n22. RobinsonANelEDonaldPSchaafH\n2007\nSA Family Practice\n49\n14\n23. MadhiSAKuwandaLSaarinenLCutlandCMothupiR\n2005\nImmunogenicity and effectiveness of Haemophilus influenzae type b conjugate vaccine in HIV infected and uninfected African children.\nVaccine\n23\n5517\n5525\n16107294\n24. MadhiSAPetersenKKhoosalMHuebnerREMbelleN\n2002\nReduced effectiveness of Haemophilus influenzae type b conjugate vaccine in children with a high prevalence of human immunodeficiency virus type 1 infection.\nPediatric Infectious Disease Journal\n21\n315\n321\n12075763\n25. von GottbergAde GouveiaLMadhiSAdu PlessisMQuanV\n2006\nImpact of conjugate Haemophilus influenzae type b (Hib) vaccine introduction in South Africa.\nBulletin of the World Health Organization\n84\n811\n818\n17128361\n26. KingJVinkPFarleyJParksMSmilieM\n1996\nSafety and immunogenicity of 3 doses of a 5-valent pneumococcal conjugate vaccine (PCV) in HIV and non-HIV-infected children <2 yrs of age.\nPediatric Research\n39\n1037\n27. AbzugMJPeltonSISongLYFentonTLevinMJ\n2006\nImmunogenicity, safety, and predictors of response after a pneumococcal conjugate and pneumococcal polysaccharide vaccine series in human immunodeficiency virus-infected children receiving highly active antiretroviral therapy.\nPediatric Infectious Disease Journal\n25\n920\n929\n17006288\n28. MadhiSAAdrianPKuwandaLJassatWJonesS\n2007\nLong-term immunogenicity and efficacy of a 9-valent conjugate pneumococcal vaccine in human immunodeficient virus infected and non-infected children in the absence of a booster dose of vaccine.\nVaccine\n25\n2451\n2457\n17023095"
+}

batch_11/PMC2533299.json

@@ -0,0 +1,4 @@
+{
+    "id": "PMC2533299",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2533299\nAUTHORS: Dimitrios Vavilis, Dimitrios Tsolakidis, Dimitrios Athanatos, Antonios Goutzioulis, John N Bontis\n\nABSTRACT:\nInversion of the uterus through the uterine lower segment incision during a caesarean section is an extremely rare obstetric incident. It consists, though, an emergency complication that is potentially life-threatening, especially in cases of prolonged inversion, because haemodynamic instability and shock may occur. Prompt diagnosis and immediate uterine reversion are the key actions in the management of this serious complication.\n\nBODY:\nIntroductionInversion of the uterus through the uterine incision during cesarean section is a rare, but potentially life-threatening emergency. When repositioning of the uterus is not immediate, excessive bleeding can cause haemodynamic instability and shock, that need proper resuscitation. To the best of our knowledge no more than 13 reports of this intraoperative complication have been found in the literature [1-13].We present a case of uterine inversion during caesarean section, that was managed successfully. It is noted that this is the first case in our department reported the last 15 years.Case presentationA 29-year-old healthy gravida two, para one with previous caesarean section underwent a lower segment caesarean section at 36 weeks for premature labor under general anaesthesia.Patient history details were as follows: Occupation: housewife; Ethnicity: Greek; Weight: 87 Kgr; Height: 167 cm; Medical history: one previous caesarean section, otherwise unremarkable; Family history: unremarkable; Patient habits and medication: non-smoker, no alcohol consuption, vitamin supplement during pregnancy.Delivery of the fetus was uneventful. After the baby was born, an intravenous bolus of 10 i.u of oxytocin was administered. Uterine contraction was noted and gentle cord traction was applied in order to remove the placenta. With slight cord traction, complete inversion of the uterus, through the uterine incision occurred with the placenta remaining firmly attached to the uterine fundus (Fig. 1). The inverted uterus was exteriorized at once and the placenta was manually removed. Several attempts for uterine reversion were done unsuccessfully for less than five minutes. Eventually, sevoflurane anesthesia was deepened from 1% to 5% and reversion of the uterus was finally achieved by gradually rolling the lowermost part of the posterior edge over the uterine fundus, thereby reverting that part that inverted last. The uterus was repositioned intraabdominally and an infusion of 20 i.u oxytocin plus 0.2 mg methylergometrine in 1000 ml Ringer's Lactated set maintained the uterine contractions. Sevoflurane progressively was reduced to the initial concentration. Uterine closure was followed by closure of the abdominal cavity. No significant changes in the haemodynamic status of the patient were noted during the operation. Blood loss was estimated at 1500 ml and two units of whole blood were transfused. After the end of cesarean section, 0.8 mg of misoprostol was given per rectum. The postoperative period was uneventful and the patient was discharged from the hospital on the 4th postoperative day.Figure 1Complete uterine inversion with fundally implanted placenta (Top: fundus of the uterus, bottom: cervical contraction ring).DiscussionThe exact incidence of uterine inversion during caesarean delivery in not known, but it seems to be an extremely rare complication. It is reported that the incidence is much lower than that of uterine inversion following vaginal delivery and occurs in approximately one out of 1860 caesarean sections [1], but we believe that this is overestimated because it is the first case reported in our department the last years. Remarkably noted that from the few cases reported in the literature there are two unique incidents of cervical inversion [13] and uterine torsion of an inverted uterus [11] during caesarean section.The causes of this complication remain unclear. Fundal insertion of the placenta [2], inherent weakness of the uterine musculature [2,3], the administration of oxytocin, in particular when given as a bolus [3,4] and traction of the cord with the placenta, either partially or completely attached to the uterus (adherent placenta) [4-6], could be probable contributing factors of this complication.Uterine inversion is a serious and potentially life-threatening complication. The principal features of this complication are haemorrhage and shock. The blood loss depends on the inversion-reversion interval and can lead to serious haemodynamic instability [3,7,8]. It has been supported that hypotension and shock may be neurogenic in origin, owing to the traction on the patient's infundibulopelvic ligaments or secondary to peritoneal or broad ligament stretching [4]. However, given that the patient is under anaesthesia, either general or regional, the neurogenic element of shock should be considered as eliminated [2], so the blood loss remains the main reason for the patients' instability. Management of uterine inversion during caesarean section is usually simple, if diagnosed promptly within a few minutes. In case of a delay in diagnosis and uterus reversion, it may lead to hypotension and difficulty in repositioning the uterus, resulting possibly in fatal outcome [2,3].The administration of volatile anaesthetic agent in high concentrations, such as halothane or sevoflurane, may facilitate the rapid repositioning of the uterus producing uterine relaxation. The exact effect of those two agents is dose-dependent depression of contractility and developed tension of human myometrium [9,14].In our case controlled cord traction was followed immediately by complete inversion. The administration of bolus oxytocin, the firm fundal insertion of the placenta, and the cord traction, although controlled and gentle could have been contributing factors. Of course, an inherent uterine musculature weakness cannot be excluded, but this is extremely difficult to be proven. Anaesthesia deepening by increasing sevoflurane concentration facilitated the manual uterine reposition. Prompt diagnosis of the complication and rapid manipulation of the uterus facilitated an successful outcome.In conclusion, uterine inversion during cesarean section is a serious and often unexpected obstetric complication. The obstetrician should be aware of this complication. Prompt diagnosis and uterine reversion without any delay are the key in the management of this life-threatening obstetric emergency.ConsentWritten informed consent was obtained from both patients – in their native language – for publication of this case report and accompanying image. Copies of the written consent are available for review by the Editor-in-Chief of this journal.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsDV conceived the study and participated in patient management, acquisition of data, interpretation of data, and was a major contributor in writing the manuscript. DT participated in patient management, acquisition of data, and drafting of the manuscript. DA revised critically the manuscript adding substantial intellectual content. AG participated in patient management, acquisition of data, and drafting of the manuscript. JB coordinated the study and patient management and revised critically the manuscript. All authors have read and approved the final manuscript. The manuscript is not under consideration and has not been published by another journal.\n\nREFERENCES:\nNo References"
+}

batch_11/PMC2533304.json

@@ -0,0 +1,4 @@
+{
+    "id": "PMC2533304",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2533304\nAUTHORS: Tobias Ettl, Johannes Kleinheinz, Ravi Mehrotra, Stephan Schwarz, Torsten E Reichert, Oliver Driemel\n\nABSTRACT:\nBackgroundA cutaneous angiosarcoma is a rare malignant tumour of vascular endothelial cells with aggressive clinical behaviour and poor prognosis. Diagnosis is often delayed due to its variable and often benign clinical appearance.Case presentationThis case presents a 64-year-old man with a six-month-history of a recurrent diffuse and erythematous painless swelling below the left eye. Several resections with intraoperatively negative resection margins followed, but positive margins were repeatedly detected later on permanent sections. Histopathologic examination of the specimen diagnosed a cutaneous angiosarcoma. Neither, finally achieved negative margins on permanent sections, nor a following chemotherapy could prevent the recurrence of the disease after five months and the patient's dead 21 months after the first diagnosis.ConclusionThe case elucidates the current diagnostic and therapeutic dilemma of this entity, which shows an unfavourable clinical course in spite of multimodal therapy.\n\nBODY:\nBackgroundA cutaneous angiosarcoma (synonyms: lymphangiosarcoma and haemangiosarcoma) is a rare malignant tumour of vascular endothelial cells. It occurs predominantly in the elderly and is confined to the face and the scalp region in more than 50% of cases [1]. Despite the aggressive behaviour and poor prognosis, the diagnosis is often delayed due to its variable and often benign clinical appearance. This case documents a facial cutaneous angiosarcoma in an elderly male patient, revealing the diagnostic and therapeutic dilemma of this entity, which shows an unfavourable clinical course in spite of multimodal therapy.Case reportA 64-year-old man presented with a six month history of a recurrent diffuse and erythematous painless swelling (3 × 2 cm2) below the left eye to the Department of Dermatology, Regensburg University, Germany. Cervical lymphadenopathy was clinically not detectable. Routine laboratory results showed no abnormality. Presuming an allergic dermatitis, topical treatment with steroids was initiated. Because of the persistence of the lesion, an incisional biopsy was performed three weeks later (Figure 1). Histopathology of the specimen showed an invasively growing tumour of the dermis, composed of atypical vascular endothelia in a disordered manner, forming bizarre vascular lumina. The tumor cells were characterized by an elevated proliferated activity with a proliferation fraction (MIB-1) of 5%–10%. The vascular endothelial proliferation showed a papillary architecture accompanied by small lymphocytes. The majority of endothelial cells presented a hyperchromatic nucleus and a swollen cytoplasm. (Figure 2a, 2b, 2c). Immunohistochemical studies demonstrated positivity for CD 31 (Figure 2d) and factor VIII-related antigen. Based on these findings the diagnosis of a cutaneous angiosarcoma was made.Figure 1Clinical appearance after first incisional biopsy: Discreet skin erythema below the left eye.Figure 2Histopathology. a: Overview image: Epidermis, followed by dermis with hair follicles and sebaceous glands. Tumour with unclear borders in the depth (H&E, 16×). b: In detail: Atypic, swollen endothelial cells with anastomosing, pseudopapillar patterns and lymphocytic inflammation (H&E, 200×). c: Immunohistochemistry with proliferation marker MIB-1 indicating proliferation in about 5%–10% of the cells (MIB-1, 200×). d: Positive immunohistochemical reaction to the endothelial marker CD 31 (CD 31, 200×).After referral of the patient to the Department of Oral and Maxillofacial Surgery, Regensburg University, Germany, the tumour was removed by wide local surgical excision (Figure 3) and the defect was temporarily covered by Epigard. Despite negative intraoperative frozen section margins, positive margins were repeatedly detected later on permanent sections. Negative margins on permanent section were finally reached after three resections and infraorbital soft tissue was plastically reconstructed with a buccal rotation flap. After surgery, chemotherapy followed with six cycles of alpha-interferon.Figure 3Clinical finding after first surgery: Intraoperative defect, 4 × 2.5 cm2 in size.Five months later a periorbital redness and swelling on both sides (Figure 4) required another incisional biopsy, which was confirmed as recurrent angiosarcoma on histopathological examination. Imaging staging procedures (MRI and CT head-neck, CT chest, CT abdomen, PET and bone scan) found bone invasion to the nasal root (Figure 5). Metastases to the neck lymph nodes as well as distant metastases were clinically and radiologically excluded.Figure 4Recurrence 5 months after first surgery: Periorbital erythema and swelling on both sides (left more than right).Figure 5MRI (axial): Left infraorbital mass with infiltration to the lateral nasal root.Neither radiochemotherapy with a cumulative radiation dose of 64.8 Gy and seven cycles Doxorubicin nor an additional antiangiogenetic therapy with Trofosfamide, Pioglitazone, Rofecoxibe and steroids could prevent the rapid tumour progression. The patient died 21 months after the first diagnosis.DiscussionThere are three main types of cutaneous angiosarcoma: Idiopathic angiosarcoma of the head and neck in elderly patients, lymphoedema-associated angiosarcoma (Stewart-Treves-Syndrome) and postirradiation angiosarcoma [2]. Besides an association with persistent chronic lymphoedema, previous irradiation and pre-existing vascular malformation, little is known regarding the causative factors of that disease [3]. With respect to pathogenesis, among others, upregulation of the glykopeptide VEGF-D, a vascular endothelial growth factor, seems to be responsible for the endothelial cell proliferation [4].Clinically the appearance of a cutaneous angiosarcoma of the skin and scalp can be variable. Early lesions most commonly present as single or multifocal ill-defined, bruise-like erythematous-purplish areas with indurated borders [5]. In the present case, akin to those previously described by others [6,7], these haematoma-like lesions can be misinterpreted as benign inflammatory or allergic hyperemias. More advanced lesions can present as dark bluish, sometimes keratotic papules or nodules with ulceration and bleeding, mimicking other malignancies like squamous cell carcinoma, basal cell carcinoma, malignant melanoma, lymphoma as well as metastases [3,5,8].Microscopically a cutaneous angiosarcoma is typically characterized by numerous, irregular and anastomosing vascular channels. These are lined by pleomorphic, hyperchromatic endothelial cells with variable mitotic activity [9]. Immunhistochemical positivity for the endothelial markers CD 31 and factor VIII-related antigen as well as for the transcription factor Fli-1 may help to establish diagnosis [10,11]. The differential diagnosis includes hemangioma, especially tufted, cavernous and epithelioid hemangioma on the one hand and acantholytic carcinoma on the other hand. Especially in immunocompromised patients Kaposi-sarcoma might be a further differential diagnosis. In the current case the presence of many lymphocytes might be a hint to regard the lesion as of lymphatic vessel origin, i.e. as a lymphangiosarcoma.Treatment of the cutaneous angiosarcoma is generally based on radical surgery and postoperative radiation therapy. Surgery is postulated to attain a wide excision of the tumour with histologically negative margins [1,4]. Unfortunately achieving negative margins is difficult, as multifocal and extensive microscopic spread is common in this disease. Intraoperative frozen sections are often performed to assist in determining section margins. Pawlik et al. [5] demonstrated, however, an overall negative predictive value of only 33.3% for that procedure, which explains the repeating surgical resections in the case report. For this reason, temporary reconstruction with homografts or skin substitutes is recommended until the definite histological confirmation of margin status. Since up to 78% of the patients still have residual tumour after wide and multiple surgical resections [5,12], this goal of achieving histologically negative section margins remains debatable. In many cases the resulting extensive resection defects require large secondary plastic reconstruction.More recently, chemotherapy and gene therapy are increasingly available. Doxorubicin is reported to be active in angiosarcoma [13], but did not show response in the present patient. Paclitaxel is another agent, that seems to have substantial effects, even in patients, who were treated previously with chemotherapy or radiation therapy [2,14]. In more palliative situations, antiangiogentic therapy with pioglitazone, rofecoxib and metronomic trofosfamide has been recommended [15].ConclusionDespite multimodal therapy options, prognosis of the cutaneous angiosarcoma is still poor, with a 5-year-survival rate between 12% and 33%. About half of the patients are dying within 15 to 18 months of presentation [1,5,16]. The most important positive prognostic factors seem to be young age, small tumour size, negative resection margins and radiation therapy [3,5,8].In summary, the present case of a cutaneous angiosarcoma of the face elucidates the current diagnostic and therapeutic dilemma of this lesion. Diagnosis is often delayed, due to its putatively innocous clinical appearance. Negative microscopic section margins are hardly achieved during surgery, resulting in multiple operations with large postoperative defects. Despite multimodal therapy concepts, the prognosis remains poor.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsTE drafted the manuscript. JK helped to the critical review of the article. RM helped to the critical review of the article. SS performed the histopathological investigations. TER helped to the critical review of the manuscript. OD performed the surgical procedure, helped to draft the manuscript, helped to the critical review of the manuscript.All authors read and approved the final manuscript.Consent sectionWritten 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_11/PMC2533353.json

@@ -0,0 +1,4 @@
+{
+    "id": "PMC2533353",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2533353\nAUTHORS: Mathieu Boxus, Jean-Claude Twizere, Sébastien Legros, Jean-François Dewulf, Richard Kettmann, Luc Willems\n\nABSTRACT:\nThe Tax1 oncoprotein encoded by Human T-lymphotropic virus type I is a major determinant of viral persistence and pathogenesis. Tax1 affects a wide variety of cellular signalling pathways leading to transcriptional activation, proliferation and ultimately transformation. To carry out these functions, Tax1 interacts with and modulates activity of a number of cellular proteins. In this review, we summarize the present knowledge of the Tax1 interactome and propose a rationale for the broad range of cellular proteins identified so far.\n\nBODY:\n1 IntroductionHuman T-lymphotropic viruses (HTLV-1 to -4) belong to the Deltaretrovirus genera of the Orthoretrovirinae subfamily. HTLV-1 was the first discovered human retrovirus in the early eighties [1]. HTLV-2 was described two years later [2] whereas HTLV-3 and -4 subtypes were isolated only recently [3,4]. HTLV-1 is the etiological agent of an aggressive leukemia called adult T-cell leukemia/lymphoma (ATL) and a neurodegenerative disease, tropical spastic paraparesis/HTLV associated myelopathy (TSP/HAM). Isolated from a case of hairy-cell leukemia, HTLV-2 is by far less pathogenic although its involvement in the development of TSP has been reported [5,6]. HTLV-3 and -4 have not yet been associated to any pathology, likely due to their recent identification and to the low number of isolates. Three HTLV subtypes have closely related simian viruses (named STLV-1, -2 and -3) while a STLV-5 strain is presently still devoid of a human counterpart [7]. Another related deltaretrovirus, bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukemia. BLV infection of sheep has been used as an animal model for HTLV [8].The genome of the HTLV viruses contain typical structural and enzymatic genes (gag, prt, pol and env) flanked by two long terminal repeats (LTRs) but also harbors an additional region called pX located between the env gene and the 3'-LTR. This region contains at least four partially overlapping reading frames (ORFs) encoding accessory proteins (p12I, p13/p30II), the Rex post-transcriptional regulator (ORF III) and the Tax protein (ORF IV). The complementary strand of the HTLV-1 proviral genome is also transcribed, yielding spliced isoforms of the Hbz factor [9-11]. Hbz interacts with factors JunB, JunD, CREB and CBP/p300 to modulate gene transcription [12-14]. There is an inverse relantionship between high Hbz and low Tax expresssion in primary ATL [15].Among proteins encoded by HTLV-1, Tax1 exerts an essential role in viral transcription as well as in cell transformation [11,16-18]. These pleiotropic functions are directed by a very wide spectrum of interactions with cellular proteins. In this review, we summarize the current knowledge pertaining to the Tax1 interactome and focus more particularly on its impact on transcription, viral persistence and transformation.2 Interaction of Tax1 with transcription factors and post-transcriptional regulatorsIn eukaryotes, initiation and elongation of gene transcription requires decondensation of the locus, nucleosome remodeling, histone modifications, binding of transcriptional activators and coactivators to enhancers and promoters and recruitment of the basal transcription machinery to the core promoter [19,20]. Tax1 is a pleiotropic transcription factor that interferes with several of these mechanisms and modulates transcription of a wide range of cellular genes. In fact, Tax1 deregulates expression of more than one hundred genes [21] through interactions with transcriptional activators, basal transcription factors and proteins involved in chromatin remodeling. Moreover, Tax1 associates with proteins involved post-transcriptionnal control of mRNAs and further modulates gene expression.2.1 Transcriptional activators and repressors2.1.1 CREB/ATF factorsTax1 was initially described as an activator of LTR-directed transcription [22]. Three imperfectly conserved 21-base-pair (bp) repeat sequences called (TxRE) located in the U3 region of the LTR are required and sufficient to confer Tax1 responsiveness [23]. The TxRE element contains an octamer motif TGACG(T/A)(C/G)(T/A) that is flanked by a G stretch and a C stretch at the 5' and 3' sides, respectively [24]. Interestingly this octamer shares homology with the consensus cAMP-responsive element (CRE) 5'-TGACGTCA-3' [24]. Nevertheless, Tax1 exhibits poor affinity for DNA and does not bind directly to the TxRE element [25] but interacts with CRE-binding/activating transcription factors (CREB/ATF). In fact, Tax1 interacts in vitro with a number of proteins of the CREB/ATF family of transcription factors: CREB, CREM, ATF1, ATF2, ATF3, ATF4 (CREB2) and XBP1 (X-box-binding protein 1) [26-31]. These proteins share a common cluster of basic residues allowing DNA binding and a leucine zipper (b-Zip) domain involved in homo- and heterodimerization. Dimer formation modulates their DNA binding specificity and transcriptional activity [32]. Biochemical studies revealed that Tax1 promotes formation of a Tax1-CREB/ATF-TxRE ternary complex in vitro by interacting with the b-Zip domain of CREB/ATF factors. Mechanistically, Tax1 enhances the dimerization of CREB/ATF factors, increases their affinity for the viral CRE [33-36] and further stabilizes the ternary complex through direct contact of the GC-rich flanking sequences [37,38]. Tax1 then recruits co-activators (CBP/p300 and P/CAF) to facilitate transcriptional initiation (see 2.3.1). The ability of Tax1 to dimerize is required for efficient ternary complex formation and for optimal transactivation [39,40]. Interaction of Tax1-CREB/ATF with the LTR promoter DNA was further explored by chromatin immunoprecipitation (ChIP) [41]. In HTLV-1 infected human T-cells (SLB-1), Tax1 and a plethora of CREB/ATF factors as well as other b-Zip proteins bind to the LTR promoter, further confirming interaction in vivo. The fact that Tax1 interacts with ATFx adds another level of complexity since this factor represses Tax1-mediated LTR activation [42]. Tax1 is thus able to interact with positive as well as with negative CREB/ATF factors to modulate LTR promoter-directed activity.Tax1 also binds to CREB co-activator proteins called transducers of regulated CREB activity (TORCs). In fact, Tax1 interacts with the three members of this family (TORC1, TORC2 and TORC3) [43,44] and TORCs cooperate with Tax1 to activate the LTR in a CREB and p300-dependent manner. Thus, TORCs are thought to associate with the Tax1 ternary complex and participate to transcriptional activation.CREB/ATF members play a role in cell growth, survival and apoptosis by regulating CRE-directed gene transcription in response to environmental signals such as growth factors or stress [32,45]. Furthermore, CREB/ATF proteins also have significant impact on cancer development [45]. Depending on the cell type, Tax1 mutants deficient for CREB activation are incompetent for transformation or induction of aneuploidy [46-50]. Tax1 activates a variety of cellular genes through its interactions with CREB/ATF proteins, for example those encoding interleukin 17 or c-fos [51,52]. Conversely, Tax1 also represses expression of genes like cyclin A, p53 and c-myb by targeting CREB/ATF factors [53-55]. Transcriptomic profiling of cells expressing either a wild-type or a CREB-deficient Tax1 protein revealed several cellular genes controlled by CRE elements activated by Tax1 [50]. Among these, Sgt1 (suppressor of G2 allele of SKP1) and p97(Vcp) (valosin containing protein) have functions in spindle formation and disassembly, respectively.Together, these reports thus demonstrate that Tax1 interacts with a series of CREB/ATF factors and modulates expression of viral and cellular genes through CRE elements. The specific contribution of each CREB/ATF member in Tax1-mediated gene transcription remains unclear.2.1.2 Serum responsive factor and members of the ternary complex factorHTLV-1 infected T-cell lines expressing Tax1 display increased expression of AP1 (activator protein 1), a homo- or heterodimeric complex of Fos (c-Fos, FosB, Fra1 and Fra2) and Jun (c-Jun, JunB and JunD) [56,57]. Fos and Jun are under the transcriptional control of the serum responsive factor (SRF) in response to various stimuli such as cytokines, growth factors, stress signals and oncogenes. SRF binds to the SRF responsive element (SRE) located in the Fos/Jun promoters which contains two binding sites: a CarG box (CC(A/T)6GG) and an upstream Ets box (GGA(A/T)). Once SRF occupies the CArG box, the ternary complex factor (TCF) establishes protein interaction with SRF and subsequently binds the upstream Ets site. This complex then recruits the co-activators P/CAF and CBP/p300 to activate transcription.In reporter assays, Tax1 activates transcription of promoters under the control of SRE motifs [52,56,58] without direct binding to the DNA but through interactions with transcription factors associated with the SRF pathway. Tax1 has been shown to bind directly to SRF [59-61] and to various members of the TCF complex such as Sap1 (SRF accessory protein 1), Elk1, Spi1 (spleen focus forming virus (SFFV) proviral integration oncogene 1) and Ets1 [49,62,63]. Tax1 interaction with SRF results in increased binding of SRF to the SRE and altered site selection [64]. Once the complexes are stabilized, Tax1 recruits the co-activators CBP/p300 and P/CAF (see 2.3.1) and mediates transactivation [63].It thus appears that Tax1 activates transcription from CREB- and SRF-responsive sites through a similar mechanism which involves its interaction with transcription factors resulting in enhanced DNA binding, altered site selection and coactivator recruitment [16].2.1.3 Nuclear factors κB (NF-κB)HTLV-1 infected cells display increased expression of various cytokines and cytokine receptors such as interleukin 2 (IL2) and the α-subunit of its high-affinity receptor complex (IL2Rα) [65-68]. Induction of IL2 and IL2Rα expression is mediated by Tax1 activation of the NF-κB/Rel family of transcription factors [69,70]. By modulating expression of a wide range of genes involved in apoptosis, proliferation, immune response and inflammation, NF-κB is thought playing a central role in Tax1-mediated cell transformation [16].In mammals, the NF-κB family of transcription factors is composed of five structurally related members, RelA, RelB (p65), c-Rel, NF-κB1 (p50/p105) and NF-κB2 (p52/p100) which form various dimeric complexes that transactivate or repress target genes bearing a κB enhancer [71,72]. p105 and p100 are precursor proteins that are processed proteolytically to the mature p50 and p52 forms, respectively. These factors share a common Rel-homology domain (RHD) mediating their dimerization, DNA binding and nuclear localization. In non-activated cells, NF-κB dimers are trapped in the cytoplasm by inhibitory proteins called IκBs such as p105, p100, IκBα, IκBβ and IκBγ (C-terminal region of p105), that mask the nuclear localization signal of NF-κB factors through physical interaction [71,72]. NF-κB activation involves phosphorylation of IκB inhibitors by the IκB kinase (IKK), which triggers their ubiquitination and subsequent proteasomal degradation, resulting in nuclear translocation of NF-κB dimers [72,73].Tax1 associates with RelA, c-Rel, p50 and p52 after their translocation in the nucleus [61,74,75] but also directly recruits RelA from the cytoplasm [76,77]. After interaction with these NF-κB factors, Tax1 increases their dimerization which is essential for binding to target promoters [61,75,78]. When the complex is bound to the promoter, Tax1 recruits the CBP/p300 and PCAF co-activators [79,80], leading to transcriptional activation2.1.4 Other transcription factorsTax1 has been shown to associate with CCAAT binding proteins such as NF-YB (nuclear factor YB subunit) and C/EBPβ (CCAAT/enhancer-binding protein β) [81-83]. Through its binding to NF-YB, Tax1 activates the major histocompatibility complex class II promoter [82]. Besides, C/EBPβ acts as a transcriptional repressor by preventing Tax1 binding to the LTR [83]. On the other hand, Tax1 increases binding of C/EBPβ to and activates the IL-1β promoter [81]. It is noteworthy that C/EBP factors have been implicated in regulation of cellular proliferation and differentiation but also in tumor formation and leukemia development [84].Tax1 forms ternary complexes in vitro with Sp1 (specificity protein 1)/Egr1 (early growth response 1) [85] and Sp1/Ets1 [62], thereby participating directly in transcriptional activation of the c-sis/PDGF-B (platelet-derived growth factor B) proto-oncogene and PTHrP (parathyroid hormone-related protein) P2 promoters, respectively. Of note, PTHrP is up-regulated during immortalization of T-lymphocytes by HTLV-1 and plays a primary role in the development of humoral hypercalcemia of malignancy that occurs in the majority of patients with ATL [86,87].Tax1 further associates with nuclear respiratory factor 1 (NRF1) and activates the CXCR4 chemokine receptor promoter [88].Finally, the transcriptional repressor MSX2 (msh homeobox homolog 2) impairs Tax1 mediated transactivation through direct binding [89].2.2 Basal transcription factorsTax1 interacts with TFIIA (transcription factor II A) and with two subunits of TFIID: TBP (TATAA-binding protein) and TAFII28 (TBP-associated factor II 28) [90-92]. These basal transcription factors compose the preinitiation transcription complex responsible for the recruitment of RNA polymerase II. Owing to this interaction, Tax1 increases the binding of TBP to the TATAA site and further stimulates transcription initiation from the LTR [93].2.3 Chromatin modifying enzymesStructural variations of chromatin range from condensed heterochromatin to more open euchromatin, a process that depends on antagonistic effects between multiple protein complexes. Among the complexes affecting chromatin structure, there are those who are capable of altering the histones themselves, the histone deacetylases (HDAC), acetyltransferases (HAT), demethylases (HDM) and methyltransferases (HMT), and those that use the energy of ATP to change the structure of the nucleosome as the SWI/SNF complex [94-96]. Tax1 expression and HTLV-1 infection both reduce histone levels in T cells [97]. Moreover, Tax1 interacts directly and recruits several proteins involved in chromatin remodeling to modulate gene transcription. The involvement of Tax1-binding proteins in transcriptional activation has been primarily described in the context of the viral LTR. Nevertheless, similar mechanisms are also likely to participate in the activation of cellular promoters.2.3.1 HATsAcetylation of lysine residues located in the N-terminal tails of histone proteins by HATs is a crucial step for activation of gene transcription. Tax1 interacts with several HATs: p300, its homologous CREB binding protein (CBP) and p300/CBP associated factor (P/CAF) [98-102]. Tax1 recruits the CBP/p300 and P/CAF once the Tax1-CREB-TxRE complex is stabilized (see 2.1.1), each of which being able to enhance Tax1-mediated transactivation of a transiently transfected LTR reporter. CBP/p300 and P/CAF bind independently on different regions of Tax1 and interaction of Tax1 with these two cofactors is required for optimal transcriptional activity from transiently transfected but also stably integrated LTR reporters [101-103]. Surprisingly, P/CAF but not CBP/p300 is able to enhance transcription from the LTR independently of its HAT activity [101,103]. Tax1 mediates recruitment of CBP/p300 on reconstituted chromatin templates and facilitates transactivation in a HAT-activity dependent manner [104,105]. CBP/p300 presence at the LTR template correlates with histone H3 and H4 acetylation as well as increased binding of basal transcription factors and RNA polymerase II. ChIP analyses with HTLV-1 infected T cell lines indicate that Tax1, CBP/p300 and acetylated histone H3 and H4 are indeed associated with the LTR promoter [41,105].There is a long lasting debate about how Tax1 recruits CBP/p300 at the Tax1-CREB/ATF-TxRE complex. Phosphorylation of CREB at serine 133 by protein kinases A or C is required for CBP/p300 recruitment via physical interaction with the KIX domain [106-108]. It has long been suggested that Tax1 bypasses the requirement for CREB phosphorylation to recruit coactivators [98,100]. Nevertheless, recent reports indicate that Tax1 rather cooperates with phosphorylated CREB (pCREB) to induce transactivation [109,110]. High levels of pCREB are detected in Tax1 expressing cells and in HTLV-1-infected human T-lymphocytes [110]. Tax1 and pCREB interact simultaneously at two distinct binding sites on the KIX domain forming a very stable complex with the viral CRE [110,111]. Both CREB phosphorylation and Tax1 binding are needed for efficient interaction of full-length CBP to pCREB and subsequent transcriptional activation [112].Finally, Tax1 is able to repress the activity of some transcription factors by competitive usage of CBP, p300 and P/CAF. As mentionned above, stable complex formation between Tax1, a transcription factor (e.g. CREB or SRF) and CBP/p300 contributes to transcriptional activation. On the contrary, when Tax1 has poor affinity for a transcription factor (e.g. p53, MyoD or STAT2), it interferes with co-activator recruitment and prevents their activition [113-116]. Although controversial, this mechanism termed trans-repression could partipate to p53 inactivation in Tax1 expressing cells and HTLV-1 infected lymphocytes (for a review see [117]).2.3.2 HDACsAmong three HDACs (-1, -2 and -3) interacting with the viral LTR in HTLV infected cell lines [118], Tax1 binds directly to HDAC1. HDAC1 overexpression represses Tax1-mediated transactivation owing to its HDAC activity [119]. Nevertheless, the presence of Tax1 and HDAC1 on the viral promoter is mutually exclusive [118,120]. HDAC1 binds to the non-activated LTR and is released from the promoter through physical interaction with Tax1 allowing recruitment of co-activators and transcription initiation. Tax1 is also able to tether HDAC1 to the tyrosine phosphatase SHP1 promoter and selectively down-regulate gene expression [121].HDACs form multiprotein complexes together with DNA-histone binding proteins such as SMRT (silencing mediator for retinoid and thyroid receptor) and MBD2 (methyl-CpG-binding domain 2) that both interact with Tax1 and are involved in Tax1 transcriptional activities [122,123]. It thus seems that Tax1, through direct association with HDACs and HDAC-containing complexes is able to selectively activate or repress viral and cellular genes expression.2.3.3 HMTs and HDMsMono-, di- and tri-methylation of histone H3 at lysine 9 (H3K9) play a crucial role in structural modification of chromatin. Tax1 associates with two enzymes involved in regulation of H3K9 methylation: SUV39H1 (suppressor of variegation 3–9 homologue 1), a HMT and JMJD2A (Jumonji containing domain 2A), a HDM [124,125]. Methylated H3K9 is a hallmark of transcriptionally inactive chromatin whereas demethylation rather promotes transcriptional activation [126]. SUV39H1 interacts with Tax1 and represses Tax1-mediated transactivation of the LTR [124]. JMJD2A is highly expressed in HTLV-1 infected cell lines but its role on Tax1-mediated transcription is currently unknown [125].Methylation of histone H3 at arginine residues is another important regulatory mechanism of transcriptionnal regulation. Tax1 associates with coactivator-associated arginine methyltransferase (CARM1), which preferentially induces methylation at residues R2, R17 and R26 of histone H3 [127]. CARM1 is recruited by Tax1 to the LTR and increases Tax1-mediated transactivation of the LTR. Consistently, silencing of CARM1 impairs Tax1 transcriptional activation, R2-, R17- and R26-methylated histone H3 proteins being present on the LTR promoter in HTLV-1 infected cells.Tax1 thus interacts with different histone methyltranferases and demethylases to modulate histone methylation and regulate gene expression.2.3.4 The SWI/SNF complexThe SWI/SNF (Switch/Sucrose Non Fermentable) complex utilizes the energy of ATP hydrolysis to remodel chromatin structures, thereby allowing transcription factors to gain access to DNA during initiation and elongation steps of transcription [128,129]. Tax1 interacts with different components of SWI/SNF: BRG1, BAFs 53, 57 and 155 [130]. Overexpression and silencing of BRG1 increments and impedes Tax1 transactivation of the LTR, respectively [130]. It was first suggested that Tax1 targets BRG1/BRM downstream of RNA polymerase II in order to prevent stalling of transcription. This model was apparently contradicted by the capacity of Tax1 to efficiently activate transcription from chromosomally integrated LTR and NF-κB promoter in a BRG1/BRM deficient cell line [131]. Nevertheless, this observation does not exclude that factors of the SWI/SNF complex cooperate with Tax1 to promote gene transcription. Consistent with this idea, Tax1 cooperates with SWI/SNF complex and RNA polymerase II to promote nucleosome eviction during transactivation [132]. Histone eviction increases accessibility of DNA to transcription factors and requires activity of SWI/SNF and RNA polymerase II [128,133]. Of note, Tax1 may also impact indirectly on SWI/SNF function [134] by interaction with DNA topoisomerase I [135].Tax1 is thus able to target SWI/SNF complex components to promote nucleosome displacement and participate to transcriptional activation.2.4 Positive transcription elongation factor b (P-TEFb) and sc35The switch from initiation of transcription to elongation requires promoter clearance and phosphorylation of the RNA polymerase II carboxyl-terminal domain (CTD) [19]. Phosphorylaton of CTD on serine 5 (S5) and 2 (S2) requires the kinase activities of the basal transcription factor TFIIH and CDK9, respectively. In the cell, CDK9 together with regulatory subunits cyclin T1, -T2, or -K compose the positive transcription elongation factor b (P-TEFb) that ensures the elongation phase of transcription by RNA polymerase II [136,137]. Tax1 recruits P-TEFb to the viral promoter by interacting with cyclin T1 and CDK9 silencing or depletion inhibits Tax1-mediated transactivation [138,139]. In fact, recruitment of P-TEFb activity to the LTR promoter increases CTD phosphorylation at serine S2 (but not S5) and allows transcriptional activation [138].Recent data suggest that the splicing factor sc35 has a critical role in P-TEFb recruitment and positively impacts on transcription [140]. Tax1 binds and colocalizes with sc35 and P-TEFb in nuclear transcriptional hot spots termed speckled structures [141].2.5 Nuclear receptorsNuclear receptors (NR) belong to a large family of ligand-activated transcription factors that regulate gene expression in response to steroids, retinoids, and other signaling molecules [142]. Tax1 functions as a general transcriptional repressor of nuclear receptors such as glucocorticoid receptors (GR) [143]. A Tax1-binding protein referred to as Tax1BP1 and identified in a yeast two hybrid screen acts as a transcriptional co-activator for NR. Tax1 represses GR signaling by dissociating Tax1BP1 from the receptor-protein containing complex. Consistently, Tax1BP1 overexpression restores GR signaling in Tax1-expressing cells [144].2.6 Post-transcriptional and translational regulatorsTax1-directed gene expression is further regulated at the post-transcriptional and translational levels through protein-protein interactions. Among these, Tax1 associates with TTP, Int6 and TRBP.2.6.1 Tristetraprolin (TTP)TTP belongs to a family of adenine/uridine-rich element (ARE)-binding proteins that contain tandem CCCH zinc finger RNA-binding domains [145]. TTP is therefore an important player in posttranscriptional regulation of mRNA containing ARE elements. Indeed, TTP delivers ARE-containing mRNAs in discrete cytoplasmic regions, called RNA granules, involved in regulation of translation or decay of these transcripts [146]. The repertoire of ARE-containing genes includes Tumor Necrosis Factor α (TNFα) and Granulocyte Macrophage-Colony-Stimulating Factor (GM-CSF) [145] involved in cell signaling, metabolism, cell proliferation, immune response, death, differentiation and morphogenesis [147].Tax1 interacts with TTP and redirects TTP from the cytoplasm to the nuclear compartment as well as in a region surrounding the nucleus [148]. Through its interactions with TTP, Tax1 stabilizes TNFα mRNA and indirectly increases TNFα protein expression. This observation is of importance for the cell transformation process induced by HTLV-1, because TNFα overexpression plays a central role in pathogenesis.2.6.2 Int6 and TRBPTax also binds Int6 (Integration site 6) and TRBP (TAR binding protein) that regulate translation and RNA interference, respectively. In fact, Int6 is a subunit of translation initiation factor eIF3, which regulates mRNA binding to the ribosome [149] while TRBP (TAR binding protein) is a componant of RISC (RNA-induced silencing complex) that mediates RNA interference [150]. Currently, the role of these interactions remains unclear.2.7 A global model of Tax1 transactivationMost of the data summarized in the former paragraphs relate to transcriptional activation of the LTR by Tax1 although it is likely that similar mechanisms also pertain to cellular promoters. Figure 1 recapitulates the mechanisms of transactivation: Tax1 relieves transcriptional repression through direct interaction with HDAC (i.e. HDAC1) and/or HMT (panel A). Tax1 interacts with CREB/ATF factors (CA) and enhances their binding to the LTR (panel B). When complexes are stabilized on the promoter, Tax1 recruits histone modifying enzymes and chromatin remodelers. This step affects chromatin structure and allows binding of basal transcription factors on the TATA box that is further stabilized by Tax1 interaction with TFIIA, TFIID and TBP (panel C). Once the initiation complex is formed, Tax1 recruits the P-TEFb factor, leading to CTD phosphorylation and processive elongation (panel D). Finally, interaction of Tax1 with SWI/SNF prevents stalling of transcription elongation.Figure 1Global model of Tax1 mediated transactivation. Tax1 relieves transcriptional repression of the LTR through direct interaction with HDAC (i.e. HDAC1) and/or HMT (panel A). Tax1 recruits CREB/ATF transcription factors (CA in panel B), histone modifying enzymes and chromatin remodelers (SWI/SNF, P/CAF and CBP/p300). Tax1 then allows binding of basal transcription factors on the TATA box (panel C). Once the initiation complex is formed, Tax1 recruits the P-TEFb factor, leading to CTD phosphorylation and processive elongation (panel D). Finally, interaction of Tax1 with SWI/SNF prevents stalling of transcription elongation. Adapted from [120,132,138,316].3 Tax1 interaction with proteins involved in cell signaling3.1 NF-κB signalingNF-κB can be activated by a series of stimuli such as antigens or cytokines that trigger two alternative pathways (so-called canonical and non-canonical). The canonical pathway is engaged in response to inflammatory stimuli (such as TNF-α and interleukin 1 IL-1), T-cell receptor activation or exposure to lipopolysaccharide (LPS). This pathway begins with the phosphorylation of IκB inhibitors by the IκB kinase (IKK), a complex of IKKα, IKKβ and IKKγ/NEMO (NF-κB Essential Modulator). IKK is activated by a mitogen-activated protein kinase kinase kinase (MAP3K) that phosphorylates the IKKα and IKKβ subunits. Phosphorylation of IκB inhibitors triggers their ubiquitination and subsequent degradation by the 26S proteasome, resulting in nuclear translocation of NF-κB dimers (e.g. p50/relA) [72,73]. The non-canonical pathway, which can be induced by stimuli such as CD40 ligand, involves IKKα activation upon phosphorylation by NF-κB inducing kinase (NIK). IKKα then phosphorylates the C-terminal region of p100 leading to subsequent processing of the p100/RelB complex into p52/RelB and its translocation into the nucleus [151]. Interestingly, p52/RelB and p50/RelA dimers target distinct κβ enhancers thereby activating different gene subsets.Tax1 stimulates both canonical and non-canonical pathways and constitutively activates NF-κB in HTLV-1 infected cells [152-154]. The above mentionned interactions of Tax1 with NF-κB transcription factors (see 2.1.3) only explains part of Tax1-mediated NF-κB activation since this completion of this process also requires cytoplamic events. In the canonical pathway, Tax1 associates with the IKKγ/NEMO subunit [155,156] as well as with activating upstream kinases such as MAPK/ERK kinase kinase 1 (MEKK1) and TGF-β activating kinase 1 (TAK1) [157,158] (see 3.2). Tax1 thus connects activated kinases to the IKK complex and forces the phosphorylation of IKKα and IKKβ leading to degradation of IκBα and IκBβ [155,156]. In addition, Tax1 binds directly to the IKKα and IKKβ subunits and activates their kinase activity independently of the upstream kinases [159]. Consistently, silencing of MEKK1 and TAK1 does not impair Tax1-induced NF-κB activation [160]. A third level of Tax1 interference with the canonical pathway is its direct binding to IκBs and their degradation independently of IKK phosphorylation [161,162]. Tax1 further interacts two subunits of the 20S proteasome (HsN3 and HC9), favors anchorage of p105 and accelerates its proteolysis [163]. Tax1 thus leads to IκB degradation at multiple levels, thereby allowing nuclear translocation of NF-κB independently of external stimuli. Besides, activation of the non-canonical pathway by Tax1 requires its interaction with IKKγ and p100 [152,154]. Through these interactions, Tax1 targets IKKα to p100, induces p100 processing and nuclear translocation of the p52/RelB dimer. It thus appears that IKKγ is an important Tax1 docking site for activation of both pathways.Post-translationnal modifications of IKKγ such as phosphorylation and K63 ubiquitination fine-tune NF-κB signaling [164,165] and are modulated by Tax1 through complex formation. In fact, PP2A activates the IKK complex by promoting dephosphorylation of IKKγ serine 68 [166,167]. Tax1 complexes with PP2A and IKKγ, maintaining the IKK complex in an active state that is required for activating NF-κB [168,169]. Ubiquitination is targeted by Tax1 through interaction with Ubc13 and Tax1BP1 [170,171]. Ubc13, an E2 ubiquitin-conjugating enzyme, is required for Tax1 interaction with IKKγ and subsequent NF-κB activation. Tax1BP1 participates to the formation of an ubiquitin-editing complex together with the deubiquitin enzyme (DUB) A20 and plays a pivotal role in termination of NF-κB and JNK signaling by regulating the activity of A20 [171-173]. A20 inhibits IKK activation by cleaving K63 linked polyubiquitin chains on tumor necrosis factor receptor (TNFR) signaling-associated factor 6 (TRAF6), receptor interacting protein 1 (RIP1) and IKKγ [174]. By disruption of complex A20-Tax1BP1, Tax1 inactivates DUB function of A20 and prevents downregulation of IKKγ ubiquitination. Consistent with this model, IKKγ is ubiquitinated in Tax1-expressing cells and in a series of HTLV-1 infected cell lines [160,171] providing a rationale for the constitutive activation of NF-κB pathway.3.2 Mitogen-activated kinases (MAPKs)MAPKs are serine/threonine-specific protein kinases that respond to external mitogen stimuli such as growth factors, cytokines or physical stress. MAPK signaling relies on a sequential phosphorylation cascade that goes through MAP kinase kinase kinase (MAP3K) to MAP kinase kinase (MAP2K) and finally to MAPK. The MAPK family includes the extracellular signal-regulated kinase protein homologues 1 and 2 (ERK1/2), ERK5, the c-Jun N-terminal Kinase 1, 2 and 3 (JNK1/2/3) also known as stress-activated protein kinase-1 (SAPK-1), the p38 isoforms (p38α/β/δ), ERK6, ERK3/4 and ERK7/8 [175]. Tax1 interacts with two MAP3Ks: MEKK1 and TAK1 [157,158].3.2.1 MEKK1MEKK1 primarily regulates JNK and ERK1/2 but also contributes to the NF-κB pathway [176,177]. Tax1 binds to the amino terminus of MEKK1 and stimulates MEKK1 kinase activity [157]. As a result, Tax1 expression increases IKKβ activity, leading to phosphorylation and degradation of IκBα. Dominant negative mutants of both IKKβ and MEKK1 prevent Tax1 activation of the NFκB pathway but, intriguingly, silencing of MEKK1 does not affect Tax1-induced NF-κB activation [160].3.2.2 TAK1TAK1 is involved in JNK, TGF-β and NF-κB dependent signaling pathways [178]. TAK1 acts in concert with TAK1 binding proteins (TABs) which link TAK1 to the upstream activating TNF receptor associated factor (TRAFs) proteins. TAK1 phosphorylates IKKβ and MKK6, thereby activating NF-κB and JNK [179].TAK1 is constitutively activated in Tax1-expressing cells and in HTLV-1 infected lymphocytes [158,160,180]. Tax1 activates TAK1 through complexation with TAK1 and TAB2 and connects TAK1 onto the IKK complex thereby stimulating IKK activity [180,181]. Consistently, overexpression of TAK1 or TAB2 increases Tax1 transactivation of a NF-κB reporter [180,181]. However, RNA interference of TAK1 suppresses activation of JNK and p38 but not NF-κB. Constitutive activation of TAK1 is thus not absolutely required for NF-κB activation [160,180]. TAK1 rather participates to JNK signaling, which is constitutively activated in Tax1-expressing cells, in Tax1-transformed murine fibroblasts and in human lymphocytes transformed with HTLV-1 [182-185].3.3 GPS-2By linking the nuclear co-receptor (NCoR)-HDAC3 complex to intracellular JNK signaling, G protein pathway suppressor 2 (GPS2) suppresses Ras/MAPK signaling and JNK1 activation [183,186,187]. Indeed, the NCoR-HDAC3 deacetylase activity represses transcription of genes involved in JNK signaling [187]. Through interaction with GPS2, Tax1 potently inhibits GPS2-mediated inactivation of JNK signaling [183]. Tax1 thus targets multiple proteins (i.e. TAK1 and GPS2) to constitutively activate JNK signaling.3.4 GTP-binding proteinsThe guanine nucleotide-binding proteins GTPases are molecular switches that cycle between active (GTP-bound) and inactive (GDP-bound) states. The G protein family includes Ras-related GTPases (or small GTPases) and heterotrimeric G proteins (α, β and γ subunits) that are activated by G protein-coupled receptors.3.4.1 Rho GTPases and the cytoskeleton proteinsTax1 complexes with several members of the small GTPase Rho family such as RhoA, Rac, Gap1m and Cdc42 [130]. Rho GTPases are activated in response to external stimuli (e.g. growth factor, stress, cytokines) and exert a wide range of biochemical functions like cytoskeleton organization, regulation of enzymatic activities as well as gene expression [188]. Notably, Tax1 binds to proteins involved in cytoskeleton structure and dynamics: α-internexin, cytokeratin, actin, gelsolin, annexin and γ-tubulin [130,189-191]. Through these interactions, Tax1 might thus connect Rho GTPases to their targets and affect cytoskeleton organization. Consistent with this idea, Tax1 localizes around the microtubule organization center (MTOC) and in the cell-cell contact region [192]. Thereby, Tax1 provides an intracellular signal that synergizes with ICAM1 engagement to cause the T-cell microtubule polarization and formation of the virological synapse. Through the formation of complexes with both Rho GTPases and their targets, Tax1 could thus favor HTLV-1 cell-to-cell transmission.Since Rho GTPases modulate a wide range of signaling networks (SRF, JNK, p38 and NF-kB) [188], complex formation with Tax1 is also likely to modulate transcription.3.4.2 Heterotrimeric Gβ subunitHeterotrimeric G proteins are the molecular switches that turn on intracellular signaling cascades in response to activation of G protein coupled receptor (GPCR). After binding of an agonist, the activated GPCR induces an exchange of GDP to GTP on the Gα subunit and facilitates the dissociation of GTP-bound Gβγ and Gα subunits [193]. Through its interaction with Gβ, Tax1 affects SDF-1 dependent activation of the CXCR4 GPCR chemokine receptor. Tax1 enhances response to SDF-1 resulting in MAPK pathway over-activation and increased cell chemotaxis. The HTLV-1 associated pathologies (ATL, HAM/TSP and dermatitis) are characterized by invasion and accumulation of infected T-cells in organs such as lymph nodes, central nervous system or dermis [194]. These results thus provide a rationale for the mechanisms of cell migration observed in HTLV-1 associated pathologies.3.5 Phosphatidylinositol 3-kinase and AP-1Phosphatidylinositol 3-kinase (PI3K) and its downstream effector Akt play a pivotal role in regulation of nutrient metabolism, cell survival, motility, proliferation and apoptosis. The PI3K family comprises eight members divided into three classes according to their sequence homology and substrate preference [195,196]. PI3K activation results in phosphorylation of Akt at Ser473 which in turn triggers a broad range of regulatory proteins and transcription factors like AP1 [197].PI3K-Akt is activated in Tax1-transformed murine fibroblasts and is required for cell transformation [198]. Tax1 complexes with the p85α regulatory subunit of PI3K [199] and inhibits activity of the p110α catalytic protein. p85α/p110α belong to the class IA PI3Ks and are activated by receptor tyrosine kinases, by Ras and Rho family GTPases and by Gβγ subunits from heterotrimeric G-proteins [200]. Since monomeric p110 is unstable and is rapidly degraded, activation of p85α/p110α does not involve the complex dissociation but would rather depend on conformational changes [201,202]. Tax1 targets p85α and disrupts the p85α/p110α complex leading to increased PI3K activity [203], Akt Ser473 phosphorylation, AP1 activation and ultimately cell proliferation. Consistent with this model, ATL cells display constitutive activation of AP1 [199,204,205].3.6 Smad proteinsTransforming growth factor β (TGFβ) inhibits T cell growth in mid-G1 but can also promote tumorigenesis [206]. TGFβ binds to a heterodimeric complex composed of type I (TβRI) and type II (TβRII) serine/threonine kinase receptors [207]. Upon binding of a TGFβ ligand, TβRII recruits and activates TβRI, which, in turn, phosphorylates downstream targets such as Smad proteins (Smad1-2-3-5-8, receptor activated R-Smad). Common mediator Co-Smad (Smad4) containing complexes then translocate to the nucleus and activate transcription of genes under the control of a Smad-binding element. Signal termination is further mediated by inhibitory Smad (I-Smad) Smad6 and Smad7 [207].Due to constitutive AP1 activation, ATL cells produce high levels of TGFβ in the sera of HTLV-1 infected patients [208]. TGFβ does not inhibit growth of HTLV-1 infected CD4+ cells but affects CD8-dependent response a mechanism that may impact on immune surveillance [209]. Furthermore, TGFβ stimulates cell surface expression of proteins involved in HTLV binding and fusion (Glut1), leading to enhanced virus transmission and production [210,211].Tax1 inhibits Smad-dependent signaling, thereby promoting resistance of HTLV-1 infected cells to TGFβ [184,212,213]. This inhibition is mediated by Tax1 interaction with the aminoterminus of Smad2, Smad3, and Smad4 [212]. Through these interactions, Tax1 inhibits complexation and DNA binding of Smad3-Smad4 [184,212]. Furthermore, Tax1 may compete with Smads for the recruitment of CBP/p300 [213].3.7 Cas-L and p130CasProteins belonging to Crk-associated substrate (Cas) family are multiadaptator and scaffold molecules that spatially and temporally control signal transduction downstream of integrins, receptors protein tyrosine kinase, estrogen receptors and GPCRs. Upon binding of a ligand to these receptors, Cas proteins are tyrosine phosphorylated and recruit adaptors and effectors (such as small GTPase) to activate downstream targets such as JNK and ERK. As a result, Cas proteins regulate cell survival, apoptosis and migration. Furthermore, deregulation of Cas functions has been linked to cell transformation, invasion and cancer [214].Among Cas proteins, Tax1 associates with p130Cas and CasL (lymphocyte type) [215]. CasL, which is preferentially expressed in lymphocytes [216], is phosphorylated and over-expressed in Tax1-expressing cells, in Tax1-transgenic mice as well as in primary lymphocytes isolated from ATL patients [215,217]. The Tax1 and CasL interplay results in enhanced motility of Tax1-expressing lymphocytes in response to fibronectin and CD3 [215]. Since CasL also participates in RhoGTPase activation, Tax1 could interconnect cytoskeleton proteins, stimulate cytoskeleton rearrangement and enhance the motility of leukemic cells.3.8 Global effects of Tax1-mediated deregulation of cell signaling pathwaysAs schematized on figure 2, Tax1 interactions with a series of components of several signaling pathways (MAPK, JNK, NF-κB, G proteins, AP1 and TGFβ) affect multiple cellular processes among which cellular activation, proliferation, cytoskeleton rearrangement, cell migration and formation of the virological synapse.Figure 2Overview of cell signaling proteins targeted by Tax1. Tax1 interacts with components of several signaling pathways (MAPK, JNK, NF-κB, AP-1 and TGF-β) and promotes cellular activation, proliferation, cytoskeleton rearrangement, cell migration and formation of the virological synapse.4 Interaction of Tax1 with cell cycle associated proteins4.1 Cyclin D-CDK4/6 complexes, Rb and CDK inhibitorsCell cycle progression is a tightly regulated process controlled by cyclins associated with cyclin-dependent kinases (CDK). Cyclins D and E cooperate with CDK4/6 and CDK2 to mediate passage through G1 phase and G1/S transition, respectively [218]. Cyclin D-CDK4/6 and Cyclin E-CDK2 complexes target the Rb retinoblastoma protein (Figure 3). In its hypophosphorylated form, Rb is bound to the transcription factor E2F1, and upon phosphorylation, Rb frees E2F1, which activates transcription of genes required for transition from G1 to S. G1/S progression can be inhibited by CDK inhibitors (CDKI) such as p15INK4b, p16INK4a, p18INK4c and p19INK4d by preventing cyclin D/CDK4/6 complex formation. Tax1 reprograms cell cycle progression, particularly at G1/S transition, through different mechanisms pertaining to transcriptional activation or repression, post-translational modifications and protein-protein interactions [219,220].Figure 3Effect of Tax1 on cell cycle progression. Through a series of interactions with cell-cycle associated proteins, Tax1 accelerates G1/S transition (A), attenuates Chk1/2 activity (B), induces supernumerary centrosomes and impedes SAC (spindle assembly checkpoint) activity (C).Tax1 is able to interact with cyclins-D1, -D2 and -D3 as well as with CDK4 and CDK6 but not with CDK1 or CDK2 [221-224]. Through these interactions, Tax1 stabilizes the cyclin D2/CDK4 complex and enhances its kinase activity, leading to hyperphosphorylation of retinoblastoma protein (Rb). Tax1 also associates with p15INK4b and p16INK4a and counteracts their inhibitory activity of CDK4 [225-228]. Finally, Tax1 binds to and targets Rb for proteosomal degradation [229]. Consistently, HTLV-1 infected cell lines and freshly isolated ATL cells display decreased levels of Rb protein.Figure 3A illustrates Tax1 interactions with components of the cyclin D/CDK complexes and provides a mechanistic model for increased G1-S phase transition efficiency as well as the accelerated cell proliferation measured in vivo [230,231].4.2 DNA repair pathway associated proteinsDNA insults and replication stress activate the DNA damage response (DDR) pathway in S and G2/M phases of the cell cycle. Activation of the DDR pathway leads to cell cycle delay or even apoptosis of severely damaged cells, and activates the DNA repair pathway. ATM (Ataxiatelangiectasia mutated) and ATR (ATM-Rad3) proteins and their respective downstream targets Chk2 (checkpoint kinase 2) and Chk1 (checkpoint kinase 1) proteins play a central role in the DDR pathway [232]. In mammals, Chk1 and Chk2 regulate Cdc25, Wee1 and p53 that ultimately inactivate CDKs which inhibit cell-cycle progression. Double-strand breaks usually activate the ATM/Chk2-dependent pathway whereas ATR/Chk1 responds to a wide variety of lesions and replication blocks [233,234].Tax1-expressing and ATL cells display DNA damages suggesting that Tax1 abrogates cellular checkpoint and DNA repair [235-237]. Tax1 binds Rad51 [130] and DNA Topoisomerase 1 [135] that are both directly involved in DNA repair processes [232,238]. Moreover, Tax1 associates and colocalizes with Chk1 and Chk2 proteins [239-242]. De novo Tax1 expression causes phosphorylation of Chk2 resulting in accumulation of cells in S-G2/M [239,243]. However, upon gamma irradiation, Tax1 inhibits Chk1 and Chk2 kinase activities and attenuates G2/M arrest and apoptosis, respectively [240,241]. Tax1 thus activates and represses checkpoint controls depending on the experimental conditions (figure 3B). In fact, Tax1 sequesters phosphorylated Chk2 within chromatin after gamma irradiation-induced DNA-damage [242]. Tax1 thereby impedes phosphorylated Chk2 chromatin egress, a mechanism required for further signal amplification and transmission [244]. Tax1 thus targets multiple components of DNA damage repair pathway and promotes DNA abnormalities.4.3 Centrosome associated proteins and spindle assembly checkpointOne of the hallmarks of Tax1-expressing cells particularly in ATL is chromosomal instability and severe aneuploidy [235], suggesting that mechanisms monitoring chromosomal segregation during mitosis are subverted by Tax1. Tax1 interacts with 4 proteins involved in centrosome amplification or in mitotic spindle assembly checkpoint (SAC) (Figure 3C).4.3.1 RanBP1 and Tax1BP2The presence of two centrosomes at mitosis is crucial for formation of bipolar mitotic spindles and correct chromosome segregation. Multipolar mitosis which happens when more than two centrosomes emerge in one cell is a possible cause of aneuploidy in solid tumors and leukemias [245]. Supernumerary centrosomes are observed in approximately 30% of ATL cells [246-248]. Tax1 colocalizes with the centrosome during mitosis and causes centrosome amplification through physical interaction with Ran/Ran Binding protein 1 (RanBP1) and Tax1BP2 [249,250]. RanBP1 is involved in the Ran GTP cycle that controls microtubule nucleation and/or stabilization and centrosome cohesion during mitosis [251,252]. Centrosome fragmentation requires direct Tax1/RanBP1 interaction and Tax1's ability to transactivate NF-κB. Tax1BP2 is thought to act as an intrinsic block to centrosome overreplication [253]. Consistently, overexpression of Tax1BP2 abolishes Tax1-induced centrosome amplification. On the other hand, a Tax1 mutant unable to bind to Tax1BP2 is impaired in centrosome overreplication [250].4.3.2 Mad1 and cdc20In eukaryotes, the mitotic spindle assembly checkpoint (SAC) monitors the fidelity of chromosome segregation [254]. SAC functioning requires complex formation between Mad1-2-3 and Bub1-2-3 proteins that arrest mitosis in response to microtubule damage [255]. At the molecular level, SAC activation involves formation of inhibitory complexes between Mad2 and/or Mad3/BubR1 and Cdc20, preventing Cdc20 from activating the anaphase promoting complex/cyclosome (APC/C). APC/C is active during mitosis where it mediates ubiquitination and degradation of an inhibitory chaperone of separase called securin. Once liberated from its inhibitor, separase triggers anaphase by hydrolysing cohesin leading to subsequent separation of sister chromatin. Furthermore, APC/C regulates the degradation of mitotic cyclin, activates CDK1 and, ultimately, promotes mitotic exit [256].Through physical interactions with Mad1 and Cdc20, Tax1 subverts activation of SAC and APC/C. Tax1 inhibits Mad1 homodimerization, a process that is required for formation of a inhibitory complex between Mad2 and Cdc20 [257-259]. Consistently, ATL cells exhibit a defect in the mitotic spindle assembly checkpoint [257]. On the other hand, Tax1 associates with and activates Cdc20-associated APC/C leading to unscheduled degradation of securin and cyclin B1, a delay or failure in mitotic entry and progression, and faulty chromosome transmission [260,261]. Tax1-induced premature activation of APC/C provokes permanent G1 arrest and senescence [262,263]. Finally, coexpression of Tax1 and securin enhances chromosomal instability and favors cell transformation in vitro and in vivo [264].5 Interaction of Tax1 with PDZ-containing proteinsThe PSD-95/Drosophila Discs Large/Zona Occludens-I (PDZ) domain containing proteins form signaling complexes at the inner surface of the cell membrane and are involved in a very broad range of functions like cell signaling, adhesion, tight-junction integrity, molecular scaffolding for protein complexes and tumor suppression [265-267]. Numerous PDZ proteins have been shown to form a complex with Tax1 owing to its PDZ binding motif (PBM) located in the C-terminus (ETEV) [268]: Pro-IL16 (precursor of interleukin 16) [269], hDLG (Drosophila Discs Large) [270,271], PSD-95, beta-syntrophin, lin-7 [268], Tip1 (Tax1 Interaction protein 1) [272], MAGI3 (Membrane Associated Guanylate kinase with inverted orientation 3) [273], hTid1 [274] and hScrib [275]. Interaction of Tax1 with these PDZ proteins frequently leads to their delocalization [273,275,276]. Functionally, PDZ proteins such as hTid1 and hScrib participate to Tax1-mediated activation of NF-κB and NFAT pathways, respectively [274,275].A Tax1-binding protein, hDLG, has been particularly studied owing to its ability to act as a tumor suppressor. hDLG acts downstream of the Wnt/frizzled pathway and binds to the adenomatous polyposis complex (APC) which mediates cell cycle progression [277,278]. APC-hDLG complex formation negatively regulates G1 to S transition and plays an important role in transducing the APC cell cycle blocking signal [277]. Besides, hDLG is also involved in maintenance and modulation of T cell polarity [279]. Through PBM/PDZ domain interaction, Tax1 induces hyperphosphorylation of hDLG, affects its localization [276] and prevents its binding to APC [271]. Interestingly, hDLG inactivation increases the ability of Tax1 to transform a mouse T-cell line [280].The Tax1 PBM is critically involved in transformation of rat fibroblasts and IL2 independent growth of mouse lymphocytes [276,281] and to promote virus-mediated T-cell proliferation in vitro and persistence in vivo [282]. In contrast, HTLV-2 Tax2 protein which does not harbor a PBM has a lower transforming activity than Tax1 [283].6 Tax1 interaction with nuclear pore and secretory pathway proteinsTax1 shuttles between the cytoplasm and the nucleus by virtue of a nuclear localization sequence (NLS) and a nuclear export signal (NES) [284-286]. In the nucleus, Tax1 is primarily located in interchromatin granules or spliceosomal speckles [141]. In the cytoplasm, Tax1 localizes to organelles associated with the cellular secretory process including the endoplasmic reticulum and Golgi complex [192,287]. Tax1 is also secreted in the supernatant of HTLV-1 infected cells isolated from HAM-TSP patients [287-289] and may behave as an extracellular cytokine. Tax1 shuttling is mediated through interaction with proteins involved in nuclear import, cytoplasmic export and secretory pathways [289-293].6.1 NucleoporinsNucleoporins of the nuclear pore complex (NPC) form a channel spanning the double lipid bilayer of the nuclear envelope. Nuclear pore complexes allow passive diffusion of ions and small proteins but translocation of cargoes larger than 40 kDa generally requires specific transport proteins [294]. Import of cargo proteins containing a classical NLS is mediated by the importin α/β dimer and requires metabolic energy which is provided by Ran GTP [295]. In contrast, carrier-independent translocation of proteins into the nucleus is energy independent and requires direct interactions with nucleoporins [295].Nuclear import and export of Tax1 are both carrier and energy independent but relies on the interaction between Tax1 and the p62 nucleoporin [290]. This interaction is mediated by the aminoterminal zinc-finger motif of Tax1. Consistently, mutations within this motif abolishes Tax1 interaction with p62 and nuclear import [290].6.2 Proteins involved in Tax1 nuclear export and secretionProteins containing a NES domain like Tax1 are expected to interact with the chromosome region maintenance 1 protein (CRM1), a member of the importin β family [296]. Under stress conditions (i.e. UV irradiation), Tax1 interacts with CRM1 and is exported outside of the nucleus, a mechanism that is inhibited by leptomycin B [291,292]. In the absence of stress however, leptomycin B does not alter subcellular distribution of Tax1 [286], suggesting that Tax1 is not exclusively exported through the CRM1 pathway.Tax1 nucleo-cytoplasmic shuttling and secretion is directed by associations with proteins involved in nuclear export (calreticulin, RanBP2, p97), in ER to Golgi transport (the coat proteins (COP) βCOP and COPII) and in movement from Golgi to plasma membrane (secretory carrier membrane protein 23 (SNAP23), secretory carrier membrane protein 1 and 2 (SCAMP1, SCAMP2)) [289,293,297]. Calreticulin, which is overexpressed in HTLV-1 infected cells, functions similarly to CRM1 by transporting proteins via NES interactions [293,298]. Tax1 secretion involves a secretory signal located in the C-terminal domain and requires interaction with SNAP23, SCAMP1 and COPII [289].Tax1 thus targets different cellular factors involved in protein transport to shuttle between nucleus, cytoplasm and extracellular environment.7 Binding domains in Tax1To interact with such a broad range of cellular targets, Tax1 contains multiple protein-binding domains (Figure 4). Among these, the N-terminal zinc finger motif associates with transcription factors (CREB/ATF [299], TBP [90], Ets1 [62], NF-YB [82], Egr1 [85]), cyclins [221], nucleoproteins (p62) [290], proteasome subunits [163] and phosphatase PP2A [168]. Mutations within this zinc finger affects Tax1-mediated CREB transactivation as well as subcellular localization due to the presence of a NLS [284]. A domain encompassing residues 55 to 95 regulate interaction of Tax1 with CBP/p300, Chk2 and Gβ2 [102,241,300]. The middle of Tax1 harbors a region required for dimerization, two leucine zipper-like motifs (aa 116–145 and 213–248) [39,301,302] and a NES sequence [291]. Substitutions within the first leucine zipper (such as T130A and L131S in mutant M22) affect Tax1 interactions with NF-κB [157,301], proteasome subunits [163] and PP2A [168]. Another mutation (S132A) abolishes Tax1 binding to coil-coiled domain containing proteins [303] (i.e. Mad1, Tax1BP1, Tax1BP2 and GPS2). A region located between the two leucine zippers is required for interaction with CARM-1, Chk2 and Gβ2 [127,241,300]. Amino acids 233–246, located within the second leucine zipper regulates Tax1 association with p15INK4b [228], p16INK4a [226], DNA topoisomerase [135] and IκBγ [161]. Consistently, the central region of Tax1 is indeed involved in NF-κB activation. Finally, the carboxyterminal region of Tax1 contains an activation domain (residues 289–332) [304] as well as motifs required for Tax1 localization within the Golgi (residues 312–315) and secretion (residues 330–332) [297]. The carboxyterminal domain is involved in Tax1 binding to Rb [229], PI3K [199], P/CAF [102], P-TEFb [138] and PDZ containing-proteins [268]. In particular, Tax1 mutant M47 (L319R, L320S) is impaired for interaction with P/CAF[102].Figure 4Functional regions of Tax1 and interaction domains. NLS (nuclear localization sequence), NES (nuclear export sequence), G (Golgi localization motif), S (secretion motif), LZR (leucine-zipper-like region), P (PDZ binding domain). Adapted from [130].8 ConclusionThe most intriguing point relating to the Tax1 interactome is the very high number of cellular proteins to which this viral oncogene is able to interact. Today, about 100 Tax1-binding proteins are identified (Table 1) and this number is permanently growing (see  for regular updates). Is it possible that a single protein modulates such a wide variety of functions? Are these interactions all relevant for the viral life cycle or pathogenesis? As schematized on Figure 5, the vast majority of these interactions contributes to viral or cellular gene expression and promotes infected cell proliferation or survival, required for maintaining viral load in vivo [231,305]. On the other hand, checkpoint abrogation allows proliferation of cells with DNA lesions and progressive accumulation of chromosomal abnormalities as frequently observed in ATL [220]. Even if one might entertain doubts about the biological relevance of some Tax1 partners, the Tax1 interactome as a whole likely contributes to the viral life cycle as well as to development of pathogenesis.Figure 5Overview of the Tax1 interactome.Table 1Cellular proteins interacting with Tax1Transcription and translationCell signalingATF1 [28]CasL [215]ATF2 [30]Cdc42 [130]ATF3 [27]Gap1m [130]ATF4 [29]GPS2 [183]ATFx [42]Gβ2 [300]BAF155 [130]IKKα [159]BAF53 [130]IKKβ [159]BAF57 [130]IKKγ [156]BRG1 [130]IκBα [162]C/EBPβ [81]IκBγ [161]CARM1 [127]MEKK1 [157]CREB [26]p100 [317]c-Rel [75]p105 [163]CREM [28]p130Cas [215]Cyclin T1 [138]p85α [199]DNA topoisomerase I [135]PP2A [168]Egr1 [85]Rac [130]Elk1 [63]RhoA [130]Ets1 [62]Smad2 [213]HDAC1 [119]Smad3 [213]Int6 [149]Smad4 [213]JMJD2A [125]TAB2 [181]MBD2 [123]TAK1 [158]MSX2 [89]Tax1BP1 [171]NF-YB [82]Ubc13 [170]NRF1 [88]p/CAF [101]p300/CBP [100]p50 [61]PDZ proteinsp52 [74]beta-syntrophin [268]RelA [75]hDLG [270]RPL6 [318]hScrib [275]Sap1 [63]hTid1 [274]Sc35 [141]lin7 [268]SMRT [122]MAGI3 [273]Sp1 [62]Pro-IL16 [269]Spi-1 [81]PSD95 [268]SRF [59]Tip1 [272]SUV39H1 [124]TAFII28 [91]Tax1BP1 [144]TBP [90]TransportTFIIA [92]Calreticulin [289]TORC1 [44]COPII [289]TORC2 [44]CRM1 [291]TORC3 [43]p62 [290]TRBP [150]p97 [289]TTP [148]RanBP2 [289]XBP1 [31]SCAMP1 [289]SCAMP2 [289]SNAP23 [289]Cell cycle and DNA repairβCOP [289]CDC20 [260]CDK4[221]CDK6 [221]Chk1 [240]CytoskeletonChk2 [239]Actin [130]Cyclin D1 [224]Annexin [130]Cyclin D2 [221]Cytokeratin [190]Cyclin D3 [224]Gelsolin [130]Mad1 [258]α-internexin [29]p15INK4b [228]γ-tubulin [191]p16INK4a [225]Rad51 [130]RanBP1 [249]Rb [229]ProteasomeTax1BP2 [250]HC9 [163]Topoisomerase 1 [135]HSN3 [163]Other viral oncogenes such as Kaposi's sarcoma-associated herpesvirus-encoded LANA and adenovirus E1A also interact with numbers of cellular proteins (e.g. more than 40 for E1A and 100 for LANA) [306,307]. Interestingly, some of these proteins are targeted both by Tax1 and E1A (such as ATF, CBP, p300 or Smad), indicating that similar signaling pathways are involved in distinct viral systems to achieve cell transformation. In particular, Tax1 and E1A share common properties that include regulation of transcriptional activation, chromatin remodeling, interference with p53 activity, regulation of proteasome function and cooperation with Ras in cell transformation [308].How are these different activities controlled temporarily and spatially? Additional studies are definitely required to address this point. Currently, Tax1 is known to shuttle between cytoplasm and nucleus, to form intranuclear speckles along with a series of cellular proteins (e.g. NF-κB factors [309], sc35 [141] and chk2 [239]) and to target specialized structures such as the centrosome [249,250]. Moreover, Tax1 localisation and protein interactions are altered under stress conditions [291,292].Despite numbers of attempts, Tax1 3-D crystallographic structure is intriguingly still unsolved suggesting that Tax1 adopts a rather undefined conformation. In this context, the concept of intrinsically disordered proteins (IDP) has recently emerged [310]. IDPs contrast to \"ordered\" proteins that fold into a unique and structured state, which represents a kinetically accessible and energetically favorable conformation. IDP proteins contain one or multiple disordered regions that exist as dynamic ensembles in which atom positions and backbone Ramachandran angles vary significantly with no specific equilibrium values [310]. The presence of short (< 30 residues) and long (> 30 residues) ID regions confer conformationnal flexibility thereby facilitating post-translational modifications and enabling a protein to functionally interact with many cellular partners [310,311]. Consistently, IDPs are frequently highly connected 'hubs' in the protein-protein networks [311-313]. In fact, Tax1 contains many proline (n = 40), serine (n = 25) and glycine (n = 25) residues that are known to promote disorder [310]. According to the VSL1 prediction programme (PONDR®, ), Tax1 contains multiple ID regions (n = 6) (Figure 6). In particular, Tax1 contains a long disordered region (spanning amino-acids 76 to 121), in contrast to the well structured capsid (p24), transmembrane (gp21) and surface (gp46) proteins (data not shown). Interestingly, other viral oncogenes such as HPV E6 and E7 are also predicted to contain significant intrinsic disorder [314].Figure 6Identification of disordered regions in Tax1 according to the VSL1 algorithm. (PONDR®, ). A domain encompassing residues 76 to 121 (black bar) corresponds to a long disordered region within Tax1.On the other hand, Tax1 is modified by phosphorylation, ubiquitination and sumoylation that potentially modulate its functions, localisation and interactions [76,315]. Tax1 also contains 8 cysteines that may form disulfite bonds or coordinate zinc ions and 48 leucines that are considered as order-promoting residues [310]. Tax1 thus appears as a flexible structure formed by a series of small modular domains that are relatively independent of surrounding sequences and that permits wide conformational changes depending upon its subcellular environment.We propose that, similarly to the hubs, the ID-based structure of Tax1 allows a wide variety of conformational changes enabling binding diversity and recognition of differently shaped protein partners. Flexible accommodation at various binding interfaces would then allow interaction of more structured domains such as the Tax1 zinc finger and leucine containing helices. This hypothetical model provides a rationale to the very broad range of Tax1 interacting proteins identified so far.In conclusion, the Tax1 interactome network with the associated biochemical studies reported here provides a molecular basis for understanding viral persistence and pathogenesis, paving the way for the design of compounds to antagonize its ability to mediate cell transformation.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsMB and LW collected data from the literature and wrote the paper, JT, SL and RK suggested comments, FD provided technical help. All authors read and approved the final manuscript.\n\nREFERENCES:\nNo References"
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+{
+    "id": "PMC2533396",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2533396\nAUTHORS: Deb K. Chatterjee, Kalavathy Sitaraman, Cassio Baptista, James Hartley, Thomas M. Hill, David J. Munroe\n\nABSTRACT:\nWe describe a novel, simple and low-cost protein microarray strategy wherein the microarrays are generated by printing expression ready plasmid DNAs onto slides that can be converted into protein arrays on-demand. The printed expression plasmids serve dual purposes as they not only direct the synthesis of the protein of interest; they also serve to capture the newly synthesized proteins through a high affinity DNA-protein interaction. To accomplish this we have exploited the high-affinity binding (∼3–7×10 −13 M) of E. coli Tus protein to Ter, a 20 bp DNA sequence involved in the regulation of E. coli DNA replication. In our system, each protein of interest is synthesized as a Tus fusion protein and each expression construct directing the protein synthesis contains embedded Ter DNA sequence. The embedded Ter sequence functions as a capture reagent for the newly synthesized Tus fusion protein. This “all DNA” microarray can be converted to a protein microarray on-demand without need for any additional capture reagent..\n\nBODY:\nIntroductionThe rapid development of genomic databases, bioinformatics tools, laboratory robotics and enabling technologies such as cDNA and oligonucleotide microarrays have provided new insights and understanding into biological and disease processes thru the global analysis of gene expression patterns. Continued development of high-throughput platforms, such as protein microarray technologies, are essential to furthering our understanding of protein function, quantitative proteomics, molecular interactions and protein profiling [1]–[3]. Unfortunately, inherent cost and technical limitations, including the required production of large libraries of purified proteins and long-term maintenance of array stability and integrity, have caused protein microarray development to lag behind that of DNA microarrays [2], [4]. Nevertheless, despite these limitations, several groups have demonstrated proof-of-concept and the potential of protein microarray technology [4]–[9].In an effort to address these issues, Nord et al. developed an alternative platform, termed protein microbead display, wherein proteins are captured via antigen-antibody binding as they are synthesized [10]. This technology utilizes a biotin labeled PCR product (containing a T7 promoter and a FLAG epitope in-frame with two IgG binding domains) anchored onto microbeads through streptavidin-biotin affinity binding. Anti-FLAG antibody is then immobilized onto the same microbead. The beads are incubated in a coupled cell-free transcription-translation extract to produce the targeted protein. Newly synthesized proteins are trapped via Flag peptide (antigen)-Flag antibody interaction. More recently, Ramachandran et al. [11] applied a similar antibody mediated protein anchoring technology to a microarray format. This platform employs purified expression construct DNAs arrayed onto a microscope slide via biotin-avidin interaction. The encoded inserts are fused with GST protein to produce GST-fusion proteins. The slides are simultaneously printed with polyclonal GST antibody to capture the newly synthesized GST fusion-proteins following coupled cell-free transcription-translation on the surface of the microarray. In both cases, newly synthesized proteins are captured through protein-protein (antigen-antibody) interactions. Although both technologies have been successfully employed, they also have their limitations. First, both platforms require a second protein, the antibody, to capture the synthesized fusion-protein. This antibody needs to be purified which adds to both labor and cost. Second, given that proteins (i.e. the capture antibody) need to be arrayed with the expression construct, maintaining the stability and integrity of the microarrays for extended periods of time remains an issue. We have addressed both of these issues by eliminating the need for antibody or other capture reagent to immobilize the newly synthesized proteins onto the microarray surface. In our system, the expression vector DNA not only directs the synthesis of each protein, but also serves to capture the protein at it's designated location on the microarray surface. Since only plasmid DNA is printed, array fabrication is simple and array stability is not an issue. To accomplish this we have exploited the high-affinity binding (∼3–7×10 −13 M) of E. coli Tus protein to Ter, a 20 bp DNA sequence involved in the regulation of E. coli DNA replication [12], [13]. In our system, each protein of interest is synthesized as a Tus fusion protein and each expression construct directing the protein synthesis contains embedded Ter DNA sequence. The embedded Ter sequence functions as a capture reagent for the newly synthesized Tus fusion protein.MethodsA. Construction of base microarray plasmidFor convenience, a recombinational cloning system was used (Invitrogen, Carlsbad, CA). First, a destination vector was made using Tus as the carboxy fusion partner. A modified, Tus (E47Q) with higher affinity for the Ter DNA sequence [12] was amplified from plasmid DNA by standard procedure. Oligos used for Tus amplifications were:Forward- 5′-ATT TTA GCT AGC GGA GGT GCG CGT TAC GAT CTC GTA GAC CGA CTC-3′ and Reverse 5′-TATATT CAA TTG TTA atg atg gtg atg atg gtg ATC TGC AAC ATA CAG GTG CAG CCG TGG 3′.Restriction sites NheI and MunI are indicated as bold and underlined. A six-histidine tag (small letters) was incorporated in reverse oligo so that Tus will be his-tagged for downstream identification.The PCR product was purified, digested with NheI and MunI, run on an agarose gel, and the fragment excised. The fragment was then cloned into a derivative pDest47 (Invitrogen, Carlsbad, CA) termed pDest472 that had been digested with the same enzymes to create pDest 472-Tus. Correct clones were selected by digestion and verified by sequencing.A Ter site (bold) was synthesized by annealing two complementary oligos:\nCCGGC CACTTTAGTTACAACATACTTATTAT\n\nCGATAATAAGTATGTTGTAACTAAAGTGG\nFollowing annealing, these oligos form a double stranded Ter site with ClaI and NgoMIV overhangs. The annealed oligo was cloned in pDest 472-Tus digested with NgoMIV and ClaI to create pDest Microarray TT-1. The clone was verified by sequencing. This is the base plasmid to clone any protein of interest by recombinational cloning.In addition to a wild type Ter site, a mutant Ter site was also tested for Tus fusion capture. The mutant Ter site was obtained during the course of cloning the wild-type Ter site. The sequence of the mutant was found to be:\nCACTTTAGTTACAACATATTTATT\nThe site of the mutation is underlined. It has been shown that mutation at this particular site will reduce binding affinity by almost 4-fold [11]. This position is equivalent to position 6 according to the nomenclature in the manuscript. The Ter sequence has been presented in reverse orientation [11].B. Construction of GFP fusion plasmidAs a proof-of-concept, we cloned GFP as a fusion with Tus. pEL100 contains eGFP gene in pDonr223 (Invitrogen, Carlsbad, CA). It contains a Kozak sequence upstream of ATG and no stop codon at the C-terminus. Thus, upon recombinational cloning into pDest Microarray TT-1, GFP will be fused in frame with Tus. Recombinational cloning was performed as per manufacturer's (Invitrogen, Carlsbad, CA) directions. Finally, the clone was sequenced to confirm correct insertion.C. Microarray FabricationMicroarray protein expression vectors were prepared in 3× standard saline citrate (SSC) in a 384-well plate (Genetix, Boston, MA) and arrayed on nitrocellulose coated “Fast Slides” (Schleicher & Schuell BioScience, Keene, NH) using a Microgrid II microarray robot at 50% humidity. Microarray features were printed at a spacing of 0.55mm (center to center) with 1.2mm spacing between each sub array. After printing, microarrays were baked at 80°C for 30 min. Slides were blocked with 0.1% PVP/0.05% Tween 20 for 1 h prior to expression.To confirm uniform DNA spotting, a sample slide from each print set was stained for DNA content using the IDT (Coralville, IA) Cy3-SpotQC detector oligo (9mers) diluted in 0.1% PVP/0.05% Tween 20 buffer. Following incubation, slides were washed 1× for 3 minutes in 10×SSC, 0.2% Sarkosyl, followed by a second wash in 10×SSC for one minute. After a third wash in 2×SSC, slides were dried and scanned with an Axon GenePix 4000 scanner (Figure S1).D. In situ Expression of Proteins\nIn situ expression was performed using a cell-free expression system (TNT Quick coupled transcription/translation system (Promega, Madison, WI)). In brief, 30 µl of rabbit reticulocyte lysate, supplemented with methionine, was added directly to the slide and incubated in a water bath. Expression and immobilization were carried out at 30°C for 1.5 hours followed by a 2 hour incubation at 15°C.E. Confirmation of Expression and Immobilization of Expressed ProteinsExpression of GFP-Tus protein was confirmed with a Cy3–Cy5 labeled antibody to the poly-histidine (poly-his) tag. Prior to incubation with labeled antibody, slides were blocked for 1 hr with 0.1% PVP/0.05% Tween 20. Monoclonal antibodies to poly-his, GFP (Sigma-Aldrich, St Louis, MO), and beta-globin (Novus, Littleton, CO) were labeled with fluorescent dye N-hydroxysuccinimide (NHS) ester-linked Cyanine 3 (Cy3) and Cyanine 5 (Cy5) (Amersham, Piscataway, NJ). In brief, 90 µl of antibody diluted to the concentration of 0.55 mg/ml in 0.1 M sodium bicarbonate/carbonate buffer pH 9.0 was mixed with 20 µl of 60 µM of Cy3 or Cy5 in sodium bicarbonate/carbonate buffer and incubated on ice. After reaction had proceeded for 90 minutes, 8 µl of Blocking Buffer (BD Biosciences, San Jose, CA) was added to the solution to quench the reactions and the solutions were allowed to sit for another 30 min with additional mixing approximately every 10 min. Unbound dye was removed by passing each sample through a size-exclusion chromatography spin column (sephadex G-15 (Sigma Aldrich, St. Louis, MO). Molar concentration for labeled protein and dye were calculated. The Cy5- labeled anti-his was mixed with equal amount of the Cy3-labeled anti-his and diluted in microarray hybridization buffer (0.1% PVP/0.1% Tween 20). Hybridization to the array was performed in an incubation chamber at 4°C with gentle rocking for at least 12 h. After incubation, slides were washed 3× for 5 minutes each in 10× PBS/0.05% Tween 20, followed by one wash in 10× PBS for one minute. All washes were performed at 4°C. Slides were dried and scanned on an Axon GenePix 4000 scanner (Union City, CA), and fluorescence data were collected and evaluated with the GenePix Pro 5.0 software. For the microarray imaging, the Axon GenePix 4000 scanner was set at 100% laser power and 350 PMT gain.System Designa) Plasmid DNA encoding protein of interest (POI) was constructed such that the protein of interest (POI) is fused with an E. coli protein called Tus. b) The plasmid also contains one or more Tus binding sites termed Ter. Tus protein binds the Ter DNA sequence as a monomer with very high affinity, ∼3–7×10 −13 M [11]. c) Plasmids are arrayed by a commercially available microarray printing robot. d) The POI-Tus fusion protein is synthesized on the microarray by coupled cell-free protein synthesis (either mammalian or prokaryotic). If the cocktail is derived from E. coli, it is made from a Tus minus strain. e) Affinity of the expressed POI-Tus fusion protein for the Ter sequence is significantly greater than the antigen-antibody affinity described by either Nord et al.[9] or Ramachandran et al [10].ResultsThe design of the expression construct (pDest-Microarray TT-1) and basic concept of the array platform are shown in Figure 1 and Figure S1. The basic expression vector is based on the Gateway (Invitrogen, Carlsbad, CA) recombinational cloning system making it easier to generate libraries of constructs. To validate the Tus-Ter DNA-binding protein system for the development of an in situ self assembling protein array, as well as demonstrate the specificity of Tus-Ter binding, we printed a microarray containing a set of clones in pDest-Microarray TT-1 encoding a GFP-Tus-His6 fusion-protein and a Ter site, an identical vector with a point mutation in the Ter site, and a third construct without a Ter site.10.1371/journal.pone.0003265.g001Figure 1Concept of protein microarray on demand.Anti-his antibody was labeled with Cy3 and Cy5, mixed in equimolar amounts, and hybridized to this microarray. The results are shown in Figure 2. We observed significant signal intensities (arbitrary units of 14000 and 20574 from Cy5 and Cy3 respectively) corresponding to the vector containing a wild-type Ter site (Fig. 2), confirming the expression and binding of the GFP-Tus fusion protein. In contrast for the vector without any Ter site, no significant signal was observed (arbitrary units of 0 and 2463 from Cy5 and Cy3 respectively; Fig. 2). The no-Ter/wild-type Ter signal ratio (−TER/+TER) is 0 for Cy5 and 0.12 for Cy3, consistent with significantly higher binding of the fusion protein to plasmid containing wild-type Ter as compared to plasmid without any Ter. Similarly, the vector containing a point mutation in the Ter site showed low to moderate signal (arbitrary units of 4738 and 10920 from Cy5 and Cy3 respectively; Fig. 2). The mutated-Ter/wild-type Ter signal ratio (mTER/+TER) is 0.34 for Cy5 and 0.53 for Cy3, indicating that a mutation in the Ter site results in reduced binding efficiency to the fusion protein. These data are in complete agreement with previous reports that Tus binds to the same mutated Ter with 4–6 fold lower efficiency \n[12. 13] as well as our own calculations of Tus: Ter and Tus: mutant Ter binding efficiencies and off rates (Table S1). These data demonstrate that an intact Ter sequence is necessary and sufficient for optimal binding to the Tus protein, allowing the effective binding of Tus-fusion proteins to Ter site(s) present in the vector and supporting the application of the Tus-Ter system for protein microarray fabrication.10.1371/journal.pone.0003265.g002Figure 2Exploiting the specificity of Tus:Ter interaction.Plasmid vectors encoding a green florescent protein (GFP)- TUS - poly-histidine fusion protein and a Ter sequence containing a point mutation (pMUT), a wild-type Ter sequence (pNOMut), and no Ter sequence were immobilized on the surface of a microarray, incubated in a cell-free rabbit reticulocyte transcription/translation extract, and hybridized with Cy-labeled anti-histidine antibody.In a parallel set of experiments, we have extended these findings to another DNA-binding protein system (lacI/LacO) and demonstrate that they can also function to mediate protein microarray fabrication in a similar manner (DC, CB, KS, JH, and DM; data not shown).As a more direct test of this platform, expression plasmids encoding 14 different proteins were immobilized onto the surface of a microarray. Expression from each of the constructs was confirmed by probing with a labeled antibody directed against the His-tag engineered into each construct (Fig. 3A). As expected, although an equal amount of DNA was printed for each feature, the relative amount of protein produced and retained by each construct varied modestly, presumably due to characteristic differential transcription/translation efficiencies (Fig. 3A and Figure S2). To validate that the individual targeted proteins were indeed expressed and captured at their designated location on the microarray, replicate arrays were probed with antibodies directed against the unique fusion partners specific for each construct (Fig. 3B and 3C). As shown, each of the target proteins was expressed and captured at a specific and designated location that was pre-determined by the insert encoded in the expression construct printed.10.1371/journal.pone.0003265.g003Figure 3Microarray feature expression and capture specificity.pNOMut variants encoding different proteins fused to TUS - poly-histidine were immobilized on the surface of a microarray and incubated in a cell-free rabbit reticulocyte transcription/translation extract. Duplicate microarrays were then hybridized with cy-labeled monoclonal antibodies specific for A) His, B) GFP, and C) human B-globin.DiscussionWe have developed a simple and cost effective strategy for the rapid generation of protein microarrays. Because only DNA expression constructs are printed, the inherent cost, stability, and technical limitations most commonly associated with other protein microarray strategies are eliminated. As, shown in Figure 1 and Figure S1, the microarray is fabricated by the printing of DNA expression constructs that function to not only direct the synthesis of the desired protein, but also as the ‘capture reagent’ for immobilization of the encoded protein onto the microarray surface. The ‘capture reagent’ function of the printed DNA expression constructs is mediated by the specific and high-affinity binding (∼3–7×10 −13 M) of E. coli Tus protein to Ter, a 20 bp DNA sequence. In Table S1 we show that both the specificity and the high-affinity binding, characteristic of wild-type Tus protein for Ter, is also true for the cloned versions of Tus:Ter interaction is maintained in a coupled in vitro transcription/translation system within a microarray environment. Finally, in Figure 3 we demonstrate that not only are the designated proteins specifically expressed, but that the individual expression constructs encoding each individual protein exclusively captures it's encoded protein without detectable diffusion or ‘bleeding’ to adjacent features on the microarray.These data support the utility and effectiveness of this platform as a method for the high-throughput production of low-cost protein microarrays for the study of protein-protein, protein-nucleic acid, or protein-small molecule interaction. The open format of these arrays, together with their long ‘shelf-life’ and simple low-cost printing scheme, make this a cost-effective, versatile, production friendly platform amendable to a wide variety of uses and applications.Supporting InformationFigure S1Design of expression construct and basic microarray fabrication schema(0.67 MB TIF)Click here for additional data file.Figure S2Validation of microarray printing. Different proteins fused to TUS - poly-histidine were immobilized on the surface of a microarray and stained for DNA content as described in Materials and Methods.(9.91 MB TIF)Click here for additional data file.Table S1(0.03 MB DOC)Click here for additional data file.\n\nREFERENCES:\n1. PandeyAMannM\n2000\nProteomics to study genes and genomes.\nNature\n405\n837\n846\n10866210\n2. MacBeathG\n2002\nProtein microarrays and proteomics.\nNature Gen.\n32\n526\n532\n3. DietrichHRKnollJvan den DoelLRvan DedemGWDaran-LapujadePA\n2004\nNanoarrays: a method for performing enzymatic assays.\nAnal Chem.\n76\n4112\n4117\n15253650\n4. GeH\n2000\nUPA, a universal protein array system for quantitative detection of protein–protein, protein–DNA, protein–RNA and protein–ligand interactions\nNucleic Acids Res.\n28\ne3\n10606671\n5. ArenkovPKukhtinAGemmellAVoloshchukSChupeevaV\n2000\nProtein microchips: use for immunoassay and enzymatic reactions.\nAnal. Biochem\n278\n123\n131\n10660453\n6. MacBeathGSchreiberSL\n2000\nPrinting proteins as microarrays for high-throughput function determination.\nScience\n289\n1760\n1763\n10976071\n7. ZhuHSynderM\n2003\nProtein chip technology.\nCurr. Opin.Chem. Biol.\n7\n55\n63\n12547427\n8. ZhuHBilginMBanghamRHallDCasamayorA\n2001\nGlobal analysis of protein activities using proteome chips\nScience\n293\n2101\n2105\n11474067\n9. NordOUhlénMNygrenPA\n2003\nMicrobead display of proteins by cell-free expression of anchored DNA.\nJ. Biotech.\n106\n1\n13\n10. RamachandranNHainsworthEBhullarBEisensteinSRosenB\n2004\nSelf-assembling protein microarrays.\nScience\n305\n86\n90\n15232106\n11. Coskun-AriFFHillTM\n1997\nSequence-specific interactions in the Tus-Ter complex and the effect of base pair substitutions on arrest of DNA replication in Escherichia coli.\nJ. Biol. Chem\n272\n26448\n26456\n9334221\n12. HendersonTANillesAFValjavec-GratianMHillTM\n2001\nSite-directed mutagenesis and phylogenetic comparisons of the Escherichia coli Tus protein: DNA-protein interactions alone can not account for Tus activity.\nMol Genet Genomics.\n265\n941\n953\n11523786\n13. BaptistaCMunroeD\n2006\n“Protein microarrays”\nVeenstraTYatesJ\nProteomics for Biological Discovery\nJohn Wiley & Sons, Inc\n189\n204"
+}

batch_11/PMC2533668.json

@@ -0,0 +1,4 @@
+{
+    "id": "PMC2533668",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2533668\nAUTHORS: David Perkins, Mark F Harris, Jocelyn Tan, Bettina Christl, Jane Taggart, Mahnaz Fanaian\n\nABSTRACT:\nBackgroundThe paper examines the key issues experienced in recruiting and retaining practice involvement in a large complex intervention trial in Australian General Practice.MethodsReflective notes made by research staff and telephone interviews with staff from general practices which expressed interest, took part or withdrew from a trial of a complex general practice intervention.ResultsRecruitment and retention difficulties were due to factors inherent in the demands and context of general practice, the degree of engagement of primary care organisations (Divisions of General Practice), perceived benefits by practices, the design of the trial and the timing and complexity of data collection.ConclusionThere needs to be clearer articulation to practices of the benefits of the research to participants and streamlining of the design and processes of data collection and intervention to fit in with their work practices. Ultimately deeper engagement may require additional funding and ongoing participation through practice research networks.Trial RegistrationCurrent Controlled Trials ACTRN12605000788673\n\nBODY:\nBackgroundThere is an increasing need for complex health service changes to be evaluated in general practice. This is in recognition of the critical role of general practice in the health system and the need for it to respond to new challenges such as the rise of chronic disease and the pressure faced by other sections of the health system such as hospitals. In the 1990s, the majority of funded research in Australian general practice was descriptive [1]. More recently there has been an increased emphasis on conducting randomised trials to produce the high level evidence needed to underpin quality primary health care policy and practice [2]. This has resulted in an increasing number of complex intervention studies commencing in Australian general practice over the past five years.It is established that such trials need a methodology capable of answering the questions they ask. This includes controlling for other changes which may be occurring and an adequate sample size to adjust for clustering of patients by practices and for loss to follow up [3]. Complex health interventions are built up of several components that may include organisational structures and delivery methods. The UK Medical Research Council framework on evaluating complex interventions emphasises the importance of a stepwise approach to developing and evaluating a complex intervention involving theory, modelling, piloting, and following the trial with an implementation phase [4]. Campbell et al have stressed the importance of the context in which the research is undertaken including health service systems; population characteristics and how these change over time; understanding the problem including the pathways by which problems are caused; the potential for improvement; reviewing barriers to the intervention; optimising components of the intervention and refining the target group to take into account its likelihood of responding to the intervention [5].There has been little empirical examination of these issues in general practice in Australia. This paper seeks to explore some of the issues related to the recruitment and retention of general practices in such trials using our own experience with a health services trial conducted over the past three years.MethodsThe trial grew from our previous research on the capacity of general practices to provide quality care for patients with chronic disease that showed a relationship between teamwork within the practice and quality of care [6,7]. Following this, we designed a study to examine the effects of an intervention to increase the team roles of non-GP staff in management of patients with chronic disease. This began with a qualitative study to identify the interventions appropriate for Australian general practice [8]. Focus groups were used to collect data from groups of practice staff: practice nurses, practices managers and receptionists. Semi-structured interviews were conducted by phone with key informants and Chief Executive Officers of Divisions of General Practice (Australian primary care organisations). This research highlighted the importance of key characteristics including leadership, communication protocols, team meetings, information systems and procedures, role definitions and training within the practice.The intervention consisted of a six month \"Teamwork for Chronic Disease Care\" program. Based on published evidence, the programme assisted practices to define roles and procedures for practice staff to support GPs in the care of patients with type-2 diabetes, ischaemic heart disease, or hypertension. The program included four elements:• Practices nominated a non-GP members of staff as chronic disease management (CDM) leader for the program• The researchers provided education and briefing sessions for GPs and CDM leaders on chronic disease care support, guidelines for structured care for diabetes and cardiovascular disease, and practice systems which asses quality and cost effective teamwork in chronic disease care• Practice visits by a teamwork facilitator to help practices assess their existing systems, set targets for change, explore barriers and enablers to team working and quality improvement activities using non-GP staff, and addressing processes for improving practice income through providing quality care• Providing ongoing support through telephone calls and follow-up visits.The intervention was piloted in one practice before the project began. However, although the same data collection methods and instruments had been used in our previous study, there was no piloting of the full recruitment, data collection and intervention prior to commencing the study. The University of New South Wales Human Research Ethics Committee approval required arms-length recruitment of both practices and, in particular, patients. Practices could only be approached by Divisions of General Practice to participate and patients could only be approached by their practices. This meant that the researchers could not directly approach either practices or patients. The researchers asked Divisions of General Practice to seek expressions of interest from practices in their territory that passed these to the researchers. Patients who met the inclusion criteria were randomly identified by the practice and approached by mail for their consent. Only when consent was received did the researchers contact patients directly. The study protocol is summarised in figure 1. [see Additional file 1]Figure 1Summary of the \"Teamwork\" study protocol.Methodology for this paperProject evaluation staff conducted telephone interviews with practices that initially expressed interest but did not participate and with those who did participate. We asked about their reasons for participation or non-participation. Practices that agreed to participate but subsequently withdrew after baseline data collection were also asked to give a reason for withdrawing which was recorded. These responses were analysed thematically.ResultsThe recruitmentDivisions of General Practice were relatively easy to recruit. Initially 20 Divisions expressed interest and 16 participated in the study. For them the main attraction was an interest in the subject matter as this fitted with their core activities of practice support and promoting the use of chronic disease and multidisciplinary care planning. These activities were included in national Division performance indicators. However, Divisions only recruited between one and seven practices each that went on to participate in the study not the larger numbers hoped for.Withdrawal after expressing interest155 practices expressed interest in the study. Of these 87 went on to participate by consenting to take part and providing baseline data. Explanations for not participating after expressing interest were surprisingly diverse. Patient demand and practice workload were the most frequent reasons given and often associated with loss of clinical or administrative staff. This caused practices to refocus on their core business (clinical care) with participation in research being a secondary priority.Delays between the practice expressing interest and being visited by the research team occurred for a variety of reasons – communication difficulties between Divisions, the research team and practices; logistical difficulties arranging visits; and staff turnover both in practices and the research team. These delays caused a number of practices to lose interest or to become caught up in other developments or activities.In some cases individual GPs, practice managers or nurses expressed initial interest but could not convince the rest of practice that they should participate. Some larger practices could not secure the agreement of the majority of GPs to take part. Practices faced competing demands from other activities especially preparing practice accreditation or participating in the more generously funded Primary Care Collaboratives Program.Withdrawal from the trial after providing baseline dataOf the 87 practices who participated in the initial data collection, 30 subsequently withdrew. Since the research design required completion of baseline data collection before block randomisation by Division, there were long delays for some practices between recruitment, completion of baseline data collection, block randomisation and the start of the intervention. Practices found this frustrating and it led to several withdrawals by practices that had provided baseline data.Some practices withdrew during the trial when they understood more clearly the extent of data collection required. Since the trial took place over an extended period circumstances changed within particular practices including the dissolution of one practice partnership and the loss of key supporting staff including a practice manager.Practices that remained in the trial57 practices remained in the study and provided data throughout the trial. This provided sufficient power to detect differences in the outcomes between intervention and control groups although it was less than originally hoped for. However the difficulty in recruitment and retention led to a delay in the study by 12 months markedly increasing the costs of the study.Practices remaining in the trial were more likely than those who did not participate or withdrew to see an opportunity to improve the way in which the practice managed chronic disease, to have a straightforward method of decision making within the practice, and to perceive research as important in improving the quality of care in their practice.The incentives for practices to participate were largely intrinsic and related to the opportunity to assess the quality of care in their own practice and take steps to improve teamwork. Funding was not a primary consideration and was provided only to defer the costs of practice staff time in data collection and recruitment.DiscussionThe aim of this paper was to examine the key issues experienced in recruiting and retaining practice involvement in a large complex intervention trial in Australian General Practice and draw out the lessons for the conduct of such studies.We have learnt some practical lessons for the successful conduct of research in general practice. The requirement of arms-length recruitment of practices through Divisions of General Practice contributed significantly to the difficulties of recruiting general practices and retaining them throughout the trial. This was because we only had an opportunity to explain the study directly to practices after they had been briefed by the Divisions and had expressed interest. In some cases this meant that practices did not have realistic expectations of what was involved in the study and they withdrew either before or after data collection had begun.We believe that while arms-length recruitment of patients is very appropriate, arms length recruitment of practices interferes with the early establishment of an appropriate relationship between researchers and practitioners. We, as researchers, must be more directly involved in explaining the study to potential participant practices within Divisions especially in explaining the rationale, process, extent of necessary commitments and potential benefits. It is difficult for Divisions to provide this information to practices since they are not primarily research agencies but it is crucial for recruitment and continued engagement. Providing feedback to practices through direct contact, newsletters, and local presentations within their Division were important.Partnerships with Divisions of General Practice are necessary in the Australian context because of their recognition as gatekeepers to general practice and because direct recruitment of practices is discouraged by most institutional ethics committees. If Divisions are to assist in recruitment (and possibly in the conduct of research) they need to see the research as central to their needs and those of their members. However in our experience, and that of other Australian researchers, engagement of Divisions and successful recruitment is likely to be enhanced if they are formally recognised as research partners and not simply used as a means to obtain practices [9]. We have sought to develop this partnership through better communication of findings of previous research with them and involvement of the Divisions as partners early and throughout the research development process. For intervention research, there may be particular benefits in involving Divisions in the implementation of the intervention (and of course funding them to do so). This is because they are experienced in working with their members, visiting practices, providing training and education and other practice support [10]. It may also allow research studies to more clearly separate the intervention and evaluation arms of their research.In most research trials, the engagement of practices also needs to be maintained over time. While funding may never be sufficient to act as an incentive on its own, it can reduce the costs of participation. The Australian Association for Academic General Practice has called for similar levels of remuneration to that provided to teaching practices for practices participating in research. Apart from financial measures, more rapid feedback and recognition for their involvement may make it more attractive to GPs. While attaining Continuous Professional Development (CPD) points is an important recognition for some GPs at some points in the CPD cycle, recognition of their continuing involvement in research may also be encouraged by other measures such as invitations to presentations of the findings, formal appointments as research collaborators or as research network members [11,12].We found it vitally important to minimise the time intervals between expression of interest and practice visits and between recruitment and initiating the intervention within each practice. This required a change in our research protocol to allow earlier contact with the practices and to minimise the delays due to block randomisation. We have also recognised the burden of research participation for general practice and have learnt to minimise the amount of time required of all practice staff, including GPs.We anticipate that practice recruitment to research will become more, not less, difficult in the medium term due to increasing work pressures, shortage of GPs and other primary care staff and competition for practice involvement from other initiatives and programs.Those designing and evaluating complex interventions in Australian general practice face very real constraints in recruiting and retaining sufficient practices. This is particularly so for health service intervention studies, such as our study of teamwork, that some GPs may find less attractive than specific therapeutic interventions. Research methodology must respond flexibly to the needs of and demands on practices. Random allocation of practices has methodological advantages to randomisation of individual patients or non-random allocation. However this may act as a significant barrier to engagement of practices even where delayed intervention is offered to the control group of practices. Other designs (such as quasi-experimental designs) are likely to be more acceptable and may need to be considered where they can answer the research questions.ConclusionIn this paper we have described some pragmatic and more serious obstacles to conducting intervention research studies in Australian general practices. Overcoming these obstacles is important for the following reasons. In the face of an increased burden of chronic diseases, we need to know if care is most effectively and efficiently organised. In a period of international workforce shortage, both medical, allied health and nursing we need to know how teams with an appropriate skill-mix are able to work together to provide the best possible care. The first challenge in trying to answer these questions is to engage general practice in the research process. Meeting this challenge requires all our creativity and ingenuity.AbbreviationsGP: general practitioner; CDM: Chronic disease management; Division: Division of General Practice; CPD: Continuous Professional Development.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsDP drafted the paper. JoT, BC, JaT, and MF undertook the underpinning research and MFH substantially revised the paper. All authors read and approved the final manuscript.Pre-publication historyThe pre-publication history for this paper can be accessed here:Supplementary MaterialAdditional file 1Why this paper is important.Click here for file\n\nREFERENCES:\nNo References"
+}

batch_11/PMC2533670.json

@@ -0,0 +1,4 @@
+{
+    "id": "PMC2533670",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2533670\nAUTHORS: Elisabeth Le Bihan-Duval, Martine Debut, Cécile M Berri, Nadine Sellier, Véronique Santé-Lhoutellier, Yves Jégo, Catherine Beaumont\n\nABSTRACT:\nBackgroundThe qualitative properties of the meat are of major importance for poultry breeding, since meat is now widely consumed as cuts or as processed products. The aim of this study was to evaluate the genetic parameters of several breast meat quality traits and their genetic relationships with muscle characteristics in a heavy commercial line of broilers.ResultsSignificant levels of heritability (averaging 0.3) were obtained for breast meat quality traits such as pH at 15 min post-slaughter, ultimate pH (pHu), color assessed by lightness L*, redness a* and yellowness b*, drip loss, thawing-cooking loss and shear-force. The rate of decrease in pH early post-mortem and the final pH of the meat were shown to be key factors of chicken meat quality. In particular, a decrease in the final pH led to paler, more exudative and tougher breast meat. The level of glycogen stored in breast muscle estimated by the Glycolytic Potential (GP) at slaughter time was shown to be highly heritable (h2 0.43). There was a very strong negative genetic correlation (rg) with ultimate meat pH (rg -0.97), suggesting a common genetic control for GP and pHu. While breast muscle weight was genetically positively correlated with fiber size (rg 0.76), it was negatively correlated with the level of glycogen stored in the muscle (rg -0.58), and as a consequence it was positively correlated with the final pH of the meat (rg 0.84).ConclusionThis genetic study confirmed that selection should be useful to improve meat characteristics of meat-type chickens without impairing profitability because no genetic conflict was detected between meat quality and meat quantity. Moreover, the results suggested relevant selection criteria such as ultimate pH, which is strongly related to color, water-holding capacity and texture of the meat in this heavy chicken line.\n\nBODY:\nBackgroundAs in other animal species, the technological quality of poultry meat is now of major importance, since poultry meat is nowadays usually consumed as cuts or as processed products rather than as whole carcasses. As already reported for pigs [1], technological quality refers to several meat properties, including water-holding capacity (i.e. drip loss during storage), intensity and homogeneity of color, firmness, shelf-life and processing yields. Meat quality is closely related to the decrease in muscle pH post-mortem. Rapid postmortem decline in pH (evidenced by low pH value measured 15 min post-slaughter in poultry, i.e. pH15) results in PSE (pale, soft, exudative) meat with a pale aspect and reduced water-holding capacity [2,3]. Variations in the extent of decrease in pH are also responsible for variations in meat quality. Low ultimate pH (measured 24 h post-slaughter in poultry) results in \"acid meat\", with similar defects to those of PSE meat [4], while high ultimate pH leads to DFD (dark, firm, dry) meat with dark color and poor storage quality [5]. In pigs, the PSE meat and \"acid meat\" defects have been shown to be controlled by major genes [1], i.e. halothane sensitivity [6] and RN [7,8] genes, respectively.The inclusion of meat quality in pig breeding schemes dates back to the 1970–1980s [1]. Varying emphasis has been given to traits of interest according to country such as meat color (certainly the most widely used quality indicator), pH and intramuscular fat content. Genetic studies on meat quality traits in poultry are more recent. Quite significant levels of heritability (ranging from 0.35 to 0.57) were obtained for meat pH, color and water-holding capacity in two studies conducted on the same experimental broiler line slaughtered under experimental conditions [9,10]. More moderate heritability values (ranging from 0.12 to 0.22) were reported for the same meat traits measured in turkeys slaughtered under commercial conditions [11]. A study performed in quails [12] also reported moderate to high levels of heritability (0.22–0.48) of ultimate meat pH and color indicators. The present study reports the first evaluations of genetic parameters of meat quality traits and their genetic correlations with growth and muscle characteristics in a commercial broiler line.ResultsDescriptive statistics for growth and body composition traits and for muscle and meat characteristics are summarized in Table 1. Distributions of these traits were close to normality, except for drip loss (DL) for which slight asymmetry was observed (data not shown).Table 1Descriptive statistics and heritability estimates for body weight, body composition, muscle characteristics and meat quality traits.TraitsNMean ± SDMin.Max.h2 ± SEGrowth and body compositionBody weight at 6 weeks (g)5922141 ± 326123430280.49 ± 0.06Weight gain from 4 to 6 weeks (g)5961040 ± 22935716460.30 ± 0.05Pectoralis major muscle weight (g)578148.6 �� 28.453.0231.90.38 ± 0.06Breast muscle yield (%)58017.8 ± 1.511.422.40.30 ± 0.04Abdominal fat (%)5832.6 ± 0.60.64.60.48 ± 0.06Muscle characteristicsFiber Cross Section Area (μm2)5921831 ± 42663031570.41 ± 0.06Lactate (μmol/g muscle)59633.1 ± 10.06.356.90.27 ± 0.05pH 15 min post-mortem (pH15)5996.45 ± 0.136.026.790.30 ± 0.05Glycolytic Potential (μmol/g muscle)591108.0 ± 17.770.0167.20.43 ± 0.05Ultimate pH (pHu)5875.64 ± 0.125.356.040.34 ± 0.06Meat quality traitsLightness (L*)59054.9 ± 3.042.663.70.35 ± 0.05Redness (a*)587-0.8 ± 0.7-2.81.30.25 ± 0.05Yellowness (b*)59011.8 ± 1.68.216.80.31 ± 0.06Drip loss (DL, %)5891.6 ± 1.00.06.20.26 ± 0.04Thawing-cooking loss (TCL, %)58114.6 ± 4.83.128.60.35 ± 0.05Warner Bratzler shear force (WB, N/cm2)57014.5 ± 3.05.925.50.34 ± 0.05Heritability estimatesAs shown in Table 1, the heritability for growth and body composition traits was moderate to high (estimates ranging from 0.30 to 0.49) in this pure broiler line. Muscle characteristics such as fiber cross section area (CSA) and GP exhibited high levels of heritability (over 0.40). The traits related to decrease in pH post-mortem (i.e. lactate, pH15 and pHu) and to meat quality (color, water retention, texture) were significantly heritable, with heritability values ranging from 0.25 to 0.35.Genetic correlation estimatesThis study revealed a strong genetic association between breast muscle GP and pHu, with an estimated genetic correlation of -0.97 ± 0.03. Lactate concentration and pH15 were also highly negatively correlated (rg -0.88 ± 0.05). In contrast, the rate and extent of decrease in pH appeared to be genetically independent, since pH15 and pHu exhibited a genetic correlation of -0.05 ± 0.24.As summarized in Table 2, post-mortem muscle metabolism traits were significantly genetically related to meat quality traits. In particular, pHu exhibited significant negative genetic correlation with meat lightness and yellowness (rg -0.65 ± 0.11 and -0.54 ± 0.11, respectively), and even more marked negative correlation with meat drip loss, thawing-cooking loss and Warner Bratzler shear force (rg -0.80). As expected, opposite and somewhat less pronounced genetic correlations were found between meat quality traits and muscle GP. Muscle pH15 was mainly related to lightness and drip loss of meat (rg -0.52 ± 0.10 and -0.55 ± 0.10, respectively).Table 2Estimated genetic correlations between post-mortem muscle characteristics and meat quality traits.Meat traits1Lightness (L*)Redness (a*)Yellowness (b*)Drip LossThawing-cooking lossWarner Bratzler shear forceLactate0.28 ± 0.160.36** ± 0.080.41** ± 0.110.54** ± 0.040.20 ± 0.100.36** ± 0.07pH15-0.52 ** ± 0.10-0.02 ± 0.15-0.16 ± 0.18-0.55** ± 0.10-0.19 ± 0.10-0.24 ± 0.14Glycolytic Potential0.52** ± 0.070.51** ± 0.110.60** ± 0.100.78** ± 0.040.49** ± 0.100.52** ± 0.05pHu-0.65** ± 0.11-0.35** ± 0.13-0.54** ± 0.11-0.89** ± 0.05-0.80** ± 0.10-0.81** ± 0.061 Lactate = Lactate concentration 15 min post-slaughter; pH15 = pH measured 15 min post-slaughter; pHu = Ultimate pH.** Genetic correlation is significantly different from zero (p < 0.01).Body and breast muscle weights appeared to be significantly related to fiber size, with positive genetic correlations of 0.69 ± 0.08, 0.76 ± 0.06 and 0.48 ± 0.09 between fiber CSA and weight gain (between 4 and 6 weeks), breast muscle weight and breast muscle yield, respectively. Interestingly, breast muscle weight exhibited a significantly negative genetic relationship with muscle GP (rg -0.58 ± 0.11), and in turn a positive correlation with pHu (0.84 ± 0.07). Significantly negative genetic correlations were also found between breast muscle mass and lightness (rg -0.55 ± 0.10), drip loss (-0.65 ± 0.10), thawing-cooking loss (-0.80 ± 0.06) and Warner Bratzler shear force (-0.60 ± 0.10).DiscussionFor the first time in a commercial broiler line, this study evaluated both the contribution of genetics to variations in meat quality traits and the genetic correlations with muscle characteristics such as fiber size and glycogen content. Quite significant levels of heritability were evidenced for meat properties such as thawing-cooking loss that can affect the processability of meat, and color and toughness that can influence the sensorial quality of meat. These genetic results emphasized the importance of the decrease in muscle pH post-mortem for breast meat quality in poultry. They indicated that, as for pigs [1], the final pH has an extensive effect on the water-holding capacity, color and texture of raw and cooked meat, while the early decrease in pH mainly influences the drip loss and lightness (L*) of raw meat, at least in this genotype. Selection for a lower final pH would lead to a higher incidence of pale and exudative meat that is tough after cooking and not very appropriate for industrial processing. On the other hand, selection for a higher final pH could improve the processing yield but could also affect storage and sensorial quality because of negative influences on microbial development and juiciness of the meat [5]. Ultimate pH, lightness and drip loss of meat were introduced into the French national breeding program for pigs in the 1980s, forming a combined quality index. It has been maintained constant across the generations of selection.The strong negative genetic correlation between glycogen content of breast muscle (estimated through the glycolytic potential) and ultimate pH represents a major result in the present study. The genetic control of glycolytic potential and its genetic relationships with meat quality have been more widely studied in pigs than in poultry. Genetic studies in pigs have focused on either post-mortem glycolytic potential (PMGP), as for the present study, or on in vivo glycolytic potential (IVGP) obtained from muscle biopsy on live animals (which is not yet available for the chicken). In pigs, fairly negative genetic correlations (ranging from -0.74 to -0.99) have been reported between PMGP and pHu measured on the same muscle or on different muscles with close metabolic characteristics [13]. Corresponding correlations were slightly lower when IVGP was considered [13]. Heritability values for IVGP were around 0.25 in a population of pigs without the RN- allele [13], while an average value of 0.21 was reported for pHu [1]. These genetic results together demonstrated that GP and pHu have close genetic control, and that in poultry, as in pigs, both traits can be modified by selection. In agreement with a previous genetic study in an experimental broiler line [10], the present study indicated that the rate and the extent of decrease in pH post-mortem are under the control of different genes. A similar conclusion was drawn from a selection experiment in pigs, in which a very low genetic correlation was found between IVGP and pH measured 30 min post-mortem [14]. In the chicken, the rate of decrease in pH was shown to be influenced by behavior at slaughter and hastened by struggle activity of the birds on the shackle line, especially wing flapping [15]. However, little is known to date about the influence of genetics on such behavioral traits and the implications for meat quality.By estimating the genetic correlations, this study made it possible to correlate responses on muscle and meat quality traits with selection on growth and breast development applied in meat-type chicken. These results indicated that selection for increased breast muscle mass is expected to lead to greater fiber hypertrophy, since a strong positive genetic correlation was observed between both traits. This was in agreement with previous results obtained by comparing experimental chicken lines divergently selected for growth [16], or differing in breast yield [17]. Most studies in pigs have indicated that selection for lean growth is associated with increases in both fiber size and number [18]. The extent to which fiber number can be modified to increase breast muscle mass in the chicken has still to be investigated. Our original results also indicated that (at least in this meat-type strain) selection for increased growth and breast muscle mass can be expected to reduce glycogen storage and in turn to increase ultimate breast meat pH. Similar results have been reported at the phenotype level, when experimental and commercial chicken lines selected for increased body weight and breast yield were compared to their respective unselected control lines [19]. Inverse relationships have been reported in pigs, for which carcass leanness appeared to be moderately positively correlated with muscle GP and negatively with pHu [13,1]. This suggests that physiological and genetic factors involved in the control of GP and pHu could be at least partly different between pigs and poultry.ConclusionMeat quality homogeneity has become a major concern for the poultry market. This genetic study confirmed that selection could be valuable to improve meat characteristics. The major factors contributing to meat quality were heritable, and no genetic conflict was detected between meat quality and meat quantity. Furthermore, the present results suggest that the ultimate pH of meat is a relevant selection criterion since it was strongly related to meat color, water-holding capacity and texture. More research is now needed to define the optimal breeding strategy to improve meat quality, which could be based either on classical polygenic selection or on the use of molecular markers. The first Quantitative Trait Loci (QTL) of meat quality traits were recently identified in a cross between experimental chicken lines divergently selected for growth [20]. Such research has now to be extended to commercial flocks, in order to identify effective molecular tools for selection on poultry meat quality.MethodsAnimals, Rearing and Slaughtering ConditionsThis genetic analysis was conducted on 312 male and 293 female pedigree birds, which were the progeny of 15 sires and 64 dams. The birds originated from a male grand-parent line intensively selected for growth and breast muscle yield, and currently used by Hubbard (Chateaubourg, France) to produce parent males. As described in detail by Berri et al. [21], birds were reared in two successive batches under regular conditions in a conventional poultry house at the INRA Experimental Poultry Unit (Nouzilly, France). Birds were given ad libitum access to a standard diet throughout the rearing period and were individually weighed every two weeks (i.e. at 2, 4 and 6 weeks). At 6 weeks of age and after 7 hours feed withdrawal, all the birds were slaughtered at the experimental processing plant of the INRA Experimental Poultry Unit. Before sacrificing by ventral neck cutting, birds were electrically stunned (125 Hz AC, 80 mA/bird, 5 s) in a water bath, bled for 3 min, and scalded at 51°C for 3 min. After removal of the gut, whole carcasses were air chilled (airflow of 7 m3) and stored at 2°C until the next day.Carcass and meat quality traitsBreast muscle (Pectoralis major plus minor) and abdominal fat weights were measured after carcass dissection, 1 day post-slaughter. Corresponding ratios were calculated in relation to live body weight at 6 weeks. All measurements for meat characteristics were performed on the Pectoralis major muscle. The pH at 15 min and 24 h post mortem was measured with a portable pH-meter (Model 506, Crison Instruments, SA, Spain) equipped with a xerolyte electrode. At 15 min post mortem, pH was estimated from 2 g of muscle mixed in 18 mL of a 5 mM iodoacetate solution. This method was described as a reference method by Santé and Fernandez [22]. At 24 h post mortem, the ultimate pH of meat (pHu) was recorded by direct insertion of the xerolyte electrode in the muscle. This method was adopted because of the significant correlation obtained 24 h post mortem between the direct tissue measurement of pH and the reference \"iodoacetate\" method [22]. Breast meat color was measured at 24 h post-slaughter using a Miniscan Spectrocolorimeter with the CIE L*a*b* system, where L* represents lightness, a* redness and b* yellowness. Higher L*, a* and b* values correspond to paler, redder and more yellow meat, respectively. The water-holding capacity of breast meat was estimated through drip loss (DL) measured after 2 days of storage of the fillet hung in a plastic bag and expressed as a percentage of the initial muscle weight. After DL measurement, P. major muscle was vacuum-packed and stored at -25°C. For meat texture analysis, breast muscle was thawed overnight at 4°C, cooked in a water-bath at 85°C for 15 min to an internal endpoint temperature of 70°C, and cooled in crushed ice for 20 min. The thawing-cooking loss was expressed as a percentage of the fresh muscle weight. The toughness of cooked meat was evaluated by the Warner Bratzler (WB) shear test using an Instron Universal Testing Instrument.Muscle ParametersThe P. major muscle glycogen, glucose-6-phosphate, free glucose and lactate concentrations (expressed in μmol/g of muscle) were measured according to Dalrymple and Hamm [23] from 1 g of fresh tissue taken and homogenized in 10 ml of 0.55 moles perchloric acid 15 min post mortem. Glycogen content available in breast muscle at slaughter time was estimated through the post mortem glycolytic potential (GP) according to the Monin and Sellier [24] equation:GP = 2 [(glycogen) + (glucose) + (glucose-6-phosphate)] + (lactate).GP was expressed as micromoles of lactate equivalent per gram of fresh tissue. The CSA of P. major muscle fibers was determined as described by Rémignon et al. [16] on 12 μm-thick cross sections stained with red azurobin. Mean CSA was determined on approximately 300 fibers in 3 random fields for each muscle.Genetic Parameter EstimationDescriptive statistics for the different traits were calculated by the UNIVARIATE procedure of SAS software [25]. Genetic parameters were computed by VCE4 software using multivariate analysis and the REstricted Maximum Likelihood (REML) method [26]. The following linear mixed model was used:y = X1β1 + X2β2 + Zu + εin which y is the vector of performances observed, β1and β2 the vectors of fixed effects for batch and sex, u the vector of genetic animal effects, and e the vector of residuals. X1, X2, and Z are the corresponding incidence matrices. As pedigree information was limited to the sires and dams of the birds measured for meat quality, the maternal environmental effects could not be correctly estimated in this genetic study. The analyses on growth performance excluded body weight measurements at the early ages of 2 and 4 weeks (which are known to be influenced by maternal effects) to focus on body weight at 6 weeks or weight gain expressed as the difference between body weight at 4 and 6 weeks.Authors' contributionsEBD supervised the genetic analyses and drafted the manuscript. MD supervised the experimental design and performed the genetic analyses. CMB and VSL supervised the measurements of meat and muscle characteristics. EBD, MD, CMB, VSL, NS, and CB participated in the design of the study and data collection and helped to draft the manuscript. YJ supervised the breeding of the line and the production of the pedigree chicks used for this trial, and helped to draft the manuscript. All authors read and approved the final manuscript.\n\nREFERENCES:\nNo References"
+}

batch_11/PMC2533672.json

@@ -0,0 +1,4 @@
+{
+    "id": "PMC2533672",
+    "text": "This is an academic paper. This paper has corpus identifier PMC2533672\nAUTHORS: Rolf Lood, Matthias Mörgelin, Anna Holmberg, Magnus Rasmussen, Mattias Collin\n\nABSTRACT:\nBackgroundPropionibacterium acnes is a commensal of human skin but is also known to be involved in certain diseases, such as acne vulgaris and infections of orthopaedic implants. Treatment of these conditions is complicated by increased resistance to antibiotics and/or biofilm formation of P. acnes bacteria. P. acnes can be infected by bacteriophages, but until recently little has been known about these viruses. The aim of this study was to identify and characterize inducible phages from P. acnes on a genetic and morphological basis.ResultsMore than 70% (65/92) of P. acnes isolates investigated have inducible phages, classified morphologically as Siphoviruses. The phages have a head of 55 nm in diameter and a tail of 145–155 nm in length and 9–10 nm in width. There was no difference in carriage rate of phages between P. acnes isolates from deep infections and isolates from skin. However, there was a significant lower carriage rate of phages in P. acnes biotype IB, mostly attributed to the low carriage rate of inducible phages in biotype IB isolated from deep tissue. Most phages have a strong lytic activity against all P. acnes isolates with inducible phages, but have less lytic activity against isolates that have no prophages. Phages only infected and lysed P. acnes and not other closely related propionibacteria. All phages could infect and lyse their non-induced parental host, indicating that these prophages do not confer superinfection immunity. The phages have identical protein pattern as observed on SDS-PAGE. Finally, sequencing of two phage genes encoding a putative major head protein and an amidase and showed that the phages could be divided into different groups on a genetic basis.ConclusionOur findings indicate that temperate phages are common in P. acnes, and that they are a genetically and functionally homogeneous group of Siphoviruses. The phages are specific for P. acnes and do not seem to confer superinfection immunity.\n\nBODY:\nBackgroundPropionibacterium acnes is regarded as a commensal of human skin, but is also known to be involved in different infections such as acne vulgaris [1] and infections with orthopaedic implants [2,3]. Treatment of acne vulgaris is complicated by bacterial resistance to commonly used antibiotics [4-6]. P. acnes infections of joint prostheses are probably much more common than previously thought [7], and treatment is complicated by biofilm formation on the foreign material [8]. The complete genome of a P. acnes isolate was recently sequenced by Brüggemann et al 2004 [9], showing that P. acnes possesses several putative virulence genes including hemolysins and co-hemolysins (CAMP factors) [10-12]. The sequenced genome only contains one cryptic prophage, and in general there is limited knowledge about phages from P. acnes.Bacteriophages can enter two principally different life cycles, lytic or lysogenic. In the lytic life cycle a bacteriophage attaches to the bacterial cell and injects its genetic material. This genetic material is directly replicated, early and late phage genes are transcribed, proteins are translated, new phage particles are formed, and the bacterium is ultimately lysed to release the progeny. In the lysogenic life cycle, most known phages integrate their genome into the host genome by specific attachment and recombination events. There have been reports about phages existing as extrachromosomal circular or linear plasmid prophages, as a part of their lysogenic cycle [13-15]. These so called prophages become integrated parts of the genome and are replicated together with the bacterial genome during cell division. Prophages account for much of the genetic diversity seen in bacteria and often carry genes that are beneficial for the bacteria, including toxins and other virulence factors [16-19].Almost 30 years ago, it was reported that 18% of P. acnes isolates are carriers of bacteriophages [20], but little is known about these phages and their potential impact on virulence. Studies of P. acnes phages have been undertaken to establish a phage typing system to distinguish between different types of P. acnes [21,22] as an alternative to use fermentative and serological methods [23]. Many studies on phages have been done on other propionibacteria as Propionibacterium freudenreichii mainly due to the research impact for dairy industry [24-28].Recently Farrar et al sequenced the first genome of a P. acnes lytic phage [29]. The phage was classified as a Siphovirus with a genome of 29,739 bp encoding 48 putative genes. Characterization of phages from P. acnes gives a deeper understanding of the relationship between phages and bacteria, and may eventually lead to a new therapy to treat P. acnes infections. In this study, we have induced, isolated, and characterized 65 temperate bacteriophages from different P. acnes isolates. The phages are all classified as Siphoviruses and can be divided into different groups based on dissimilarities in two genes encoding a putative major head protein and an amidase.ResultsCarriage of phagesSince not much is known about the presence and carriage rate of bacteriophages in P. acnes, we investigated this in relation to both the site of isolation (superficial or deep infections) and to biotype. To investigate if P. acnes had prophages that could be induced to enter the lytic life cycle, we stimulated 92 different P. acnes isolates (see table 1) with 2 μg/ml mitomycin C to induce prophages, followed by analysis of the plaque forming capacity of lysates on the noninduced parental isolates. Plaques were clear with well-defined edges and had a diameter of 6–7 mm. Bacteriophages could be induced in more than 70% of the isolates examined. In this study, we have used different isolates of P. acnes (Holmberg et al, unpublished) from deep tissue (AD-isolates, mainly isolated from infections of foreign material as hip prosthesis and sternal wires), and from the skin (AS-isolates, from the skin of healthy individuals). AD-isolates and AS-isolates had a carriage-rate of 70.5% respectively 70.8% (see Figure 1A).Table 1P. acnes isolates used, result of biotyping and obtained phage isolates.IsolateBiotypePhageMajor HeadAD1IA--AD2IAPAD2*EU302613AD3IB--AD4IAPAD4*EU302614AD5IAPAD5*EU302615AD6IAPAD6*EU302616AD7IIPAD7*EU302617AD8IAPAD8*EU302618AD9IAPAD9*EU302619AD10IIPAD10*EU302620AD11IAPAD11*EU302621AD12IB--AD13IIPAD13*EU302622AD14IIPAD14*EU302623AD15IA--AD16II--AD17IAPAD17*EU302624AD18II--AD19IAPAD19*EU302625AD20IIPAD20*EU302626AD21IIPAD21*EU302627AD22IAPAD22*EU302628AD23IAPAD23*EU302629AD24IIPAD24*EU302630AD25IAPAD25*EU302631AD26IB--AD27IB--AD28IAPAD28*EU302632AD29IA--AD30IAPAD30*EU302633AD31II--AD33IB--AD35IAPAD35EU302634AD36IAPAD36*EU302635AD38IAPAD38EU302636AD39IB--AD40IAPAD40*EU302637AD41IAPAD41*EU302638AD42IAPAD42*EU302639AD43IB--AD44IAPAD44EU302640AD45IAPAD45EU302641AD47IAPAD47EU302642AD48IAPAD48EU302643AS1IB--AS2IAPAS2EU302610AS3IBPAS3EU302611AS4IAPAS4EU302651AS5IA--AS6IAPAS6EU302612AS7IAPAS7EU302652AS8IIPAS8EU302653AS9IIPAS9EU302654AS10IAPAS10EU302655AS11IBPAS11EU302656AS12IIPAS12EU302657AS13IBPAS13EU302658AS14IA--AS16IBPAS16EU302659AS18IB--AS20II--AS21II--AS22IIPAS22EU302660AS23IBPAS23EU302661AS24IAPAS24EU302662AS25IAPAS25EU302663AS26IAPAS26EU302664AS27IAPAS27EU302665AS28II--AS29IIPAS29EU302666AS30IAPAS30EU302667AS31IAPAS31EU302668AS32IA--AS33IA--AS34IA--AS35IAPAS35EU302669AS37IBPAS37EU302670AS38IB--AS39IIPAS39EU302671AS40IIPAS40EU302644AS41IIPAS41EU302645AS42IIPAS42EU302646AS43IIPAS43EU302647AS44IAPAS44EU302607AS45IA--AS46IBPAS46EU302608AS47IAPAS47*EU302648AS48IA--AS49IBPAS49EU302649AS50IAPAS50*EU302609AS51IA--AS52IIPAS52EU302650* phages from which high quality TEM micrographs were obtained.Figure 1Carriage rate of phages in different groups and biotypes of P. acnes. Phages were induced with 2 μg/ml mitomycin C, lysate sterile filtered and stored for seven days to screen out unstable phages. The lysate was then applied at different concentrations to an overlay plate with the host isolate. If plaques were observed after two days, the sample was regarded positive for phages. (A) A comparison in carriage rate of inducible phages between deep isolates (AD), skin isolates (AS) and biotype IA, IB and II. (B) A comparison of carriage rate of inducible phages in biotype IB between deep isolates and isolates from skin induced by 2 μg/ml mitomycin C.Bacterial isolates that did not have any inducible phages using mitomycin C were screened for prophages using a PCR-based approach by amplification of the gene encoding a putative major head protein and recA as a positive control. Only one of the isolates, AS14, was positive in the major head PCR, indicating that only 1/9 of these isolates has phages with similarities to the known major head gene (data not shown).The carriage rate of inducible temperate phages in the different biotypes was also examined. Biotypes had previously been determined by sequencing of recA (Holmberg et al, unpublished). Biotype IA had a higher carriage rate than IB (p < 0.01), as did biotype II (p < 0.05). Since biotype IB had a lower carriage rate compared to the other two biotypes, we compared the carriage rate of phages in the isolates determined as biotype IB between deep and superficial isolates of P. acnes. The carriage rate in biotype IB was significantly higher in superficial isolates as compared with isolates from deep infections, since none of the deep tissue isolates biotyped as IB had inducible phages (p < 0.05, see Figure 1B).Bacteriophage morphology and classificationThe classification of bacteriophages is mainly based on phage morphology and the nature of the nucleic acid [30]. Though other classification systems such as sequence similarities within genes encoding structural proteins have been proposed, classification based on morphology and the nature of the nucleic acid is still the most accepted system [31]. Forty-nine of the bacteriophages were examined using negative staining and transmission electron microscopy. All examined phages have an icosahedral head of approximately 55 nm in diameter, and a tail composed of 33 segments with a total length of 145–155 nm and a width of 9–10 nm. The tail is non-contractile and appears flexible. Most phages have a visible base plate on the tail with attached spikes (see Figure 2, 3). These morphological attributes warrant classification of the phages as Siphoviridae. Thus, this morphology is identical to PA6 [29] and very similar to the P. acnes phages studied by Zierdt [32]. Also, the phages are very similar morphologically to Siphoviruses isolated from other propionibacteria [24,25,27]. This classification was further strengthen by amino acid sequence comparison of a part of a putative major head protein with other known phage proteins, using a BLASTp search against GenBank. The best hit was on gp6 from PA6, and the second and third best hit on gp7 from Mycobacterium phage Che9d and gp7 from Mycobacterium phage Halo. All these phages are classified as Siphoviridae.Figure 2Electron micrographs of bacteriophages from P. acnes. Phages were negatively stained with 0.75% uranyl formate and subjected to transmission electron microscopy. The phages have a head of approximately 55 nm in diameter, loaded with genetic material. Their tails have a size of 150 × 10 nm and are flexible and non-contractile. In the lower micrograph, PAD25 is adhering to bacterial cell debris, and two phages have lost their heads. At the attachment site between the phage and the cell debris, a base plate with attached spikes can be observed. All phages were classified as Siphoviruses based on their morphology.Figure 3P. acnes bacteriophages classified as Siphoviruses. Phages were negatively stained with 0.75% uranyl formate. All phages were classified as Siphoviruses based on their morphology. No difference in morphology could be observed between the different phages. Several of the phages have empty heads and adhere to bacterial cell debris.Phage specificityBacteriophages are generally quite restricted in their host range. There are phages that can infect over bacterial species boundaries, but some phages are species specific and in many cases also specific for certain subgroups (subspecies, serotypes or biotypes, strains) within a species. To determine how specific the isolated phages are, different P. acnes isolates were infected with 48 different phages (see additional file 1). Bacterial isolates with inducible phages were generally easy to lyse, with plaque production in nearly 99% (2278/2304) of cases. P. acnes isolate AS12 was significantly more difficult to lyse than other isolates with inducible phages, and only 73% (35/48) of the examined phages caused plaque formation. Similarly, phage PAD8 failed to lyse 19% (9/48) of the examined isolates. In P. acnes isolates without any inducible phages, we could only observe plaque formation in 30% (144/480) of the cases. We also examined the ability of phages to cause lysis in the recently sequenced P. acnes strain KPA171202 (DSM no. 16379) of biotype IB [9]. This strain was lysed only by 40% (19/48) of the phage lysates. The different phages are not specific to certain biotypes, but seem to be less lytic against biotype II in isolates with inducible phages, since 23/26 failures to infect and lyse the bacterial isolates were in isolates of biotype II.Since all of the examined phages have identical morphology, protein pattern and in most cases very high similarity in genes encoding the putative major head protein and an amidase (see Figure 4), we examined if phage host-range could be used as a possible tool to differentiate the phages. Based on the host-range analysis we choose P. acnes isolates KPA171202, AD7, AS1 and AS5 to differentiate the phages and to divide them into different groups. By using these four isolates, we could divide the phages into 9 separate groups (see Fig 5). Sixteen of the examined phages could infect and lyse all four bacterial isolates (host-range group PA I). There is a tendency that phages isolated from biotype II and from skin can infect and lyse all four bacterial isolates, and are classified as host-range group PA I. Also, the only phages that can lyse the prophage-free isolates AD26 and AD27 (PAD21, PAS7 and PAS11) are classified as belonging to the host-range group PA I. Furthermore, none of nine selected phages (PAS2, PAS10, PAS12, PAS40, PAS50, PAD9, PAD20, PAD21 and PAD42) were able to infect and lyse P. avidum, P. granulosum or P. freudenreichii (data not shown). Our results show that isolates lacking inducible temperate phages are more difficult to lyse using phages, than isolates carrying inducible temperate phages and that the phages are specific to P. acnes.Figure 4Phylogenetic trees of phages from P. acnes. A gene encoding a putative major head protein and a gene encoding a putative amidase were sequenced in nine P. acnes phages and aligned using MacVector ClustalW alignment. Phylogenetic trees were constructed using neighbor joining with best tree mode. The putative major head protein (A) was similar between all P. acnes phages examined and showed the highest similarity to Mycobacterium phage Che9d gp7, but did also have high similarity to Lactococcus phage phiLC3 MHP and Streptococcus phage SM1 gp40. If outgroups were removed (B) four separate groups of major head proteins could be observed. One group with PA6, another with PAS2 and PAS50, a third group with PAD21 and a forth group with PAS10, PAS12, PAS40, PAD9, PAD20 and PAD42. The putative amidase (C) showed similar patterning among the phages with phages PAS2 and PAS50 representing one group closely related to PA6, while the other phages PAS10, PAS12, PAS40, PAD9, PAD20, PAD21 and PAD42 formed a second group. The closest known phage protein with similarity to the putative amidase is represented by Mycobacterium phage PG1 gp49, but more related is P. acnes own amidase.Figure 5P. acnes bacteriophages host-range groups. The host-range for phages isolated from P. acnes was determined by using a bacterial overlay of different P. acnes isolates and adding phages. Four bacterial isolates (KPA171202, AD7, AS1 and AS5) were used to divide the phages into different host-range groups. Phages in host-range group PA I could infect and lyse all four isolates, PA II all except for KPA171202, PA III (AD7, AS1), PA IV (AD7), PA V (AD7, AS5), PA VI (PAD7, PAS42), PA VII (AS1, AS5), PA VIII (AS1, AS5, KPA171202) and PA IX could not infect and lyse any of the isolates used.Phylogenetic analysis of phage genes and proteinsA part of a putative major head gene was sequenced in all isolated phages. The nucleotide sequence was aligned and a phylogenetic tree was reconstructed. The phages could be divided in two distinctly divided groups with the already sequenced phage PA6 forming a third group (see additional file 2). The whole gene encoding a putative major head protein was sequenced in nine of these phages, selected based on their partial sequencing and site of isolation (AD/AS), and the amino acid sequences were aligned and a phylogenetic tree was reconstructed (see Figure 4). The P. acnes phage putative major head protein show high similarity to phages isolated from Mycobacterium, Lactococcus and Streptococcus. The already sequenced phage PA6 forms its own group, while PAD9, PAD42, PAS10, PAS12, PAS40 and PAD20 form a large group with similar sequences, even though PAD20 is slightly different. Phages PAS2 and PAS50 form a third group and PAD21 forms a fourth separated group. This pattern is similar to the pattern seen when only using partial sequencing, except that PAD21 now seems to form its own group.We further sequenced the gene encoding a putative amidase in these nine phages (see Figure 4C). The phage pattern is similar to what could be seen when aligning the major head protein. However, PA6 is more closely related to PAS2 and PAS50 in this protein, and PAD21 do not form its own group, but have high similarity to the large group of phages. The closest phage similarity is to Mycobacterium phage PG1 gp49, but more closely related is a chromosomally encoded P. acnes amidase.DiscussionWe show that more than 70% of the investigated P. acnes isolates are carriers of inducible phages, which is a significantly higher carriage rate of inducible phages than that reported by Webster et al (18%) [20] despite that similar methods were used and isolates were from both skin and deeper infections. This is most likely due to differences in the geographic origin of isolates, and may reflect a difference in P. acnes susceptibility to mitomycin C or strain acquired resistance to phages. There was no overall difference in carriage rate of inducible phages between P. acnes isolated from skin or deep infections, indicating that carriage of phage not necessary leads to an increased virulence of the host strain. Thus it appears that the phages studied here do not carry virulence factors, which is in concordance with the lack of putative virulence factors on the recently published genome of a P. acnes phage [29]. It should be noted that at present, we only have information on the presence or absence of inducible phages. However, future studies may very well reveal differences in the gene composition of phages isolated from skin and deep isolates.We found that isolates of biotype IB have significantly lower rate of inducible phages as compared to isolates of IA and II. Interestingly, none of the isolates of biotype IB in isolates from deep infections are carriers of inducible phages (0/7), while isolates from skin of biotype IB have a carriage rate of almost 73%. The low carriage rate of inducible phages in AD-isolates typed as IB is further strengthen by the fact that none of these isolates have similarities to the phage major head gene as judged by failure of amplifying this gene in these isolates (data not shown), thus indicating that these isolates do not have any inducible phages and not any prophages with similarities to known P. acnes phages. Thereby it seems like either only biotype IB without phages can cause infections, or that biotype IB loses their phages during infection. If this is the case, it is uncertain why this only happens in biotype IB and not in biotype IA and II. The lower carriage rate in biotype IB is neither due to an increased resistance to phage entrance into the bacterial cell, since biotype IB is equally sensitive to phages as the other biotypes, as judged by additional file 1. More research in this specific area is needed to understand this phenomenon.When we examined the host isolate specificity of the phages, we found that isolate AS12 was significantly more difficult to lyse than the other isolates with inducible phages. However, AS12 is still highly susceptible to PAS12, indicating that AS12 might differ in a protein or receptor essential for most phages to be able to infect and lyse the bacterial cell. Opposite to this, we found that phage PAD8 did not lyse other isolates as efficiently as other phages. This difference could be caused by the phage favouring a lysogenic state, or that the phage receptor binding to the bacterial cell is less efficient compared to other P. acnes phages. When we added phages to P. acnes isolates without inducible phages, we found that most of these isolates were resistant to phage-mediated lysis. This is not surprising, since these isolates do not have inducible phages and thereby could have a mechanism that makes them resistant against phage infection. The different phages were not specific to certain biotypes. However, most of the failures to infect and cause lysis in the bacterial isolates were in biotype II, possibly indicating that these isolates generally are more resistant to phages, or adapt more easily to them. Grouping of the phages based on host-range did not correlate with biotype, even though there is a tendency that phages from isolates of biotype II are classified as host-range group PA I, thus indicating that phages from isolates of biotype II may have broader infective capacity as compared to biotype IA and IB. The phages also seem to be specific to P. acnes isolates, since none of the examined phages were able to infect and lyse P. avidum, P. granulosum or P. freudenreichii. All phages examined were able to infect and lyse their non-induced parental host, thus indicating that the prophages do not confer superinfection immunity as many other known prophages do [33,34]. This is in concordance with the fully sequenced phage PA6 that does not seem to have a repressor-like protein [29]. This may benefit P. acnes phage evolution by more efficient gene transfer between prophages and free phages.The phages examined could be divided into three-four groups using phylogenetic analysis of the gene encoding a putative major head protein and an amidase. This differences in sequence stress the fact that the phages in the different groups very well may have other genetic dissimilarities, providing advantages for the phages. It is also obvious that several phages have identical nucleotide sequences and perhaps should be considered as subspecies to certain groups of P. acnes phages. The changes in the gene encoding the major head do not lead to a changed morphology, as seen when comparing PAS50 with the other phage micrographs (see Figure 3). When examining the phage proteins on an SDS-PAGE, all phages have identical protein patterns with four protein bands (17 kDa, 29 kDa, 52 kDa and 54 kDa, data not shown). This indicates that the phages have very similar structural proteins, even though some substitution in amino acids occur, and may reflect an evolutionary pressure to retain the structural proteins.ConclusionWe have induced, identified, and characterized 65 temperate P. acnes phages, classified as Siphoviruses on a morphological basis. These phages are species specific and do not confer superinfection immunity. These results give new insight into the relation between P. acnes and its phages, and contribute to a better understanding of the phage-host interaction.MethodsBacterial isolatesA total of 92 P. acnes isolates were used. These were divided into two groups: 44 isolates from deep infections (AD-isolates, mainly isolated from prosthesis and sternal wires) and 48 isolates from skin from healthy individuals (AS-strain), described in Holmberg et al, unpublished. All isolates from healthy individuals and AD-isolates 1–16 and 37 are isolated in Lund, Sweden. AD-isolates 17–32 and 38–48 are isolated in Örebro, Sweden, and AD-isolate 33–36 are isolated in Malmö, Sweden. The P. acnes strain KPA171202 (DSM no. 16379), P. avidum (DSM no. 4901), P. granulosum (DSM no. 20458) and P. freudenreichii subsp. freudenreichii (DSM no. 20271) were obtained from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany).Isolation of phagesP. acnes isolates were plated from frozen stocks on Tryptic Soy Broth (TSB, Bacto, Mt Pritchard, NSW, Australia) with 1.5% Agar (TSBA, Bacto), and incubated for two days at 37°C under anaerobic conditions. Isolates were inoculated in 10 ml prereduced TSB (rTSB) followed by incubation for three days. TSB was prereduced by 24 h incubation under anaerobic conditions. The cultures were diluted 1:9 in 1 ml rTSB and incubation continued for 8 h. Mitomycin C (Calbiochem EMD Biosciences, San Diego, CA, USA) was added to a final concentration of 2 μg/ml, and incubation continued overnight. Cultures were centrifugated (10 min, 1,500 g, Eppendorf Centrifuge 5415R) and sterile filtered (Millex-GP 33 mm 0.2 μm, Millipore, Billerica, MA, USA) to obtain a phage stock. The phage stock was stored at 4°C for 7 days to clear the stock from unstable phages [32]. Phages were spotted onto TSBA plates at different concentrations (1:1 – 1:10,000) after a bacterial overlay of the host P. acnes isolate had been prepared. Plates were incubated for two days at 37°C under anaerobic conditions and examined for plaques. When single plaques were observed, these were picked with a sterile scalpel and transferred to SM-buffer (20 mM Tris-HCl pH 7.5, 100 mM NaCl, 60 mM MgSO4(7H2O), 0.01% gelatine) followed by elution overnight at 4°C. Phages were propagated by spotting phages on a TSBA plate with a bacterial overlay of its host P. acnes isolate and incubated three days at 37°C under anaerobic conditions. The overlay containing all plaques were transferred to SM-buffer, eluted overnight at 4°C, followed by sterile filtration of the SM-buffer. This method generally generated a concentration of 1011–1013 pfu/ml. Phages were named after the host bacterium with a 'P' before the bacterial isolate name. Isolates were regarded as having inducible phages if infectious phages were induced using the method described above. All bacterial isolates that did not have any inducible phages were screened for prophages using a PCR-based amplification of the major head gene (primer pair MHF/MHR) and using recA amplification as a positive control.Phage host specificityForty-eight P. acnes isolates that carried inducible phages and 10 P. acnes isolates that did not carry any inducible phages using mitomycin C were plated on TSBA as an overlay assay. To each plate, 5 μl phage stock (1010 pfu/ml) from 48 different phages was added. Plates were incubated for 2 days under anaerobic conditions and examined for plaques. Nine phages (PAS2, PAS10, PAS12, PAS40, PAS50, PAD9, PAD20, PAD21 and PAD42) were also applied to P. avidum, P, granulosum and P. freudenreichii subsp. freudenreichii as an overlay assay.Electron microscopyBacteriophages in a stock concentration of 1011–1013 pfu/ml were placed on a carbon coated copper grid and negatively stained (for references see [35]). A 0.75% uranyl formate solution was obtained by dissolving 37.5 mg uranyl formate (BDH Chemicals Ltd., Poole, UK) in 5 ml boiling water, and stabilized with 5 μl 5 M NaOH. Grids were rinsed for 45 sec with 100 μl TBS and blotted off with a filter paper. The sample (5 μl) was added to the grid, left for 45 sec and blotted off with a filter paper. The sample was washed twice with two 100 μl H2O drops and blotted off after each wash with a filter paper. The sample was stained for 3 sec with 100 μl 0.75% uranyl formate and then stained for additionally 15–20 sec with 100 μl 0.75% uranyl formate. Samples were observed using a Jeol JEM 1230 transmission electron microscope operated at 60 kV accelerating voltage, and recorded with a Gatan Multiscan 791 CCD camera.Phage gene comparisonPhage lysate (10 μl) was boiled for 10 min to release phage DNA from intact phages. A PCR (Eppendorf Mastercycler personal) was run under the following conditions: 95°C 10 min, 35 cycles of 95°C 1 min, annealing temperature 1 min 30 sec and 72°C 1 min 30 sec, ending with 72°C for 10 min. Final concentration in the mixture was 1× buffer, 0.2 mM dNTP mix, 30 mU/μl Taq polymerase, 1.5 mM MgCl2 and 1 μM each of the primers. Primers used are described in table 2. All samples were run with an annealing temperature of 56.5°C except when using primer pairs PR264/PAR-2 and MHF/MHR where an annealing temperature of 54°C was used. All reagents except for the primers are from Fermentas (Vilnius, Lithuania). PCR products were washed with SpinPrep PCR Clean-up Kit (Novagen, Madison, WI, USA), sequenced using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA), and analyzed using an ABI 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Sequences were aligned using the Clustal W algorithm [36] and a phylogenetic tree was reconstructed using the MacVector v9.5.2 software package (Cary, NC, USA). The final partial major head gene nucleotide sequences correspond to nucleotides 163–484 in gp6 from P. acnes bacteriophage PA6. The phylogenetic tree was constructed using UPGMA [37] and uncorrected p-values with a bootstrap with 1000 replications. Phylogenetic trees including outgroups with protein comparisons of a putative major head and an amidase were constructed using neighbor joining and poisson-correction with a best tree mode, while phylogenetic trees without outgroups were analysed using uncorrected p-values.Table 2Primers usedNameSequence (5'-3')ReferencePR264GCAGGCAGAGTTTGACATCC[38]PAR-2GCTTCCTCATACCACTGGTCATC[39]MHFTCCTGGTTCTATGATTGGTGCGThis studyMHRCGGAGACCCCTTCGGATACACThis studyMH1FCGTTTGTGGATGCTCTTGTCAThis studyMH1RCCTTCGGATACACCTCAGTAGACAThis studyMH2FGCTCTTGGTGCTTCGATTGGTThis studyMH2RGATACCCATCAACACCACCCCThis studyAmi1FGGTTTGAATGGTGTGAAAGGTCThis studyAmi1RTTTCGGAACATTATATTTGTCACACThis studyAmi2FTATCGAGATTTGCGCGGATThis studyAmi2RACCACGAAACGACTCCGCThis studyStatistical methodsAll statistical tests were calculated using the Chi-Square test. All tests were also run with Fischer's exact test with similar results.Nucleotide sequence accession numberAll partial sequences of the gene encoding a putative major head were submitted to GenBank. Accession numbers (EU302607–EU302671) are shown in table 1. Whole sequences of the gene encoding a putative major head for phages PAS2, PAS10, PAS12, PAS40, PAS50, PAD9, PAD20, PAD21 and PAD42 have accession numbers EU784673–EU784681 and the gene encoding a putative amidase have accession numbers EU784682–EU784690.Authors' contributionsRL participated in the design of the study and performed the isolation of phages, genetic and protein based characterization of phages, host specificity observations and drafted the manuscript. Electron microscopically examinations were done by MM. AH performed the initial characterization of the P. acnes strains. MR assisted in statistical analysis and revision of the manuscript. MC designed the study and revised the manuscript. All authors read and approved the final manuscript.Supplementary MaterialAdditional file 1Host range analysis. Propionibacterium acnes isolates were plated on TSBA as an overlay assay. Phages isolated from these strains were added to each P. acnes isolate and incubated for two days at anaerobic conditions and were analyzed for plaques. A plus (+) indicates that the bacterial isolate was lysed, and the number of + indicates the quality and extent of the lysis in the bacteria. A minus (-) indicates that no plaques were observed. P. acnes strain KPA171202 is the recently sequenced P. acnes strain. Bacterial isolates AD2-AS52 have inducible phages while bacterial isolates AD1-KPA171202 do not have inducible phages.Click here for fileAdditional file 2Phylogenetic tree of P. acnes phages based on partial sequencing on a gene encoding a putative major head protein. A part of the gene encoding the putative structural protein major head protein was amplified and sequenced. Obtained nucleotide sequences were compared using MacVector ClustalW Alignment and a phylogenetic tree was constructed using UPGMA and uncorrected p-values with 1000 replications for bootstrap. The phages were divided into two distinct groups, with the recently sequenced phage PA6 forming a third group.Click here for file\n\nREFERENCES:\nNo References"
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