Methods accounting for competing risks in time-to-event problems are becoming common in mainstream statistical analyses. Standard approaches include those based on log-rank type tests  and cumuWe investigated the behaviour of the estimate of treatment effect under different (i) rates of competing risk events and (ii) censoring patterns. Through extensive simulations, we compared event-specific analyses to competing risk approaches, and also the power and type I error of different weighting schemes for common methods of analysis under these conditions. These approaches were also examined on actual data on time to relapse in patients receiving bone marrow transplant for multiple myeloma, with the competing risk being transplant-related mortality.Differences between treatment effect estimates using proportional hazards and proportional subhazards methods were evident at competing event rates as low as 2.5%, even when there was no difference in competing risks between treatment groups. For the multiple myeloma example, the p-values were 0.0003 (log-rank), 0.005  and 0.05Competing risk approaches should be included in time-to-event analyses, even when the rate of competing events is expected to be low. When comparing two groups, using weights from separate censoring distributions is recommended as this has a less inflated Type I error and greater power. This may be thought of as analogous to using separate variances when comparing two means rather than using a pooled variance, although the increased efficiency of the pooled approach does not exist here when there is no true difference in censoring risk.


Informing missing heritability for complex disease will likely require leveraging information across multiple SNPs within a gene region simultaneously to characterize gene and locus-level contributions to disease phenotypes. To this aim, we introduce a novel strategy, termed Mixed modeling of Meta-Analysis P-values (MixMAP), that draws on a principled statistical modeling framework and the vast array of summary data now available from genetic association studies, to test formally for locus level association. The primary inputs to this approach are: (a) single SNP level p-values for tests of association; and (b) the mapping of SNPs to genomic regions. The output of MixMAP is comprised of locus level estimates and tests of association. In application of MixMAP to summary data from the Global Lipids Gene Consortium, we suggest twelve new loci  for low-density lipoprotein cholesterol (LDL-C), a causal risk factor for cardiovascular disease and we also demonstrate the potential utility of MixMAP in small data settings. Overall, MixMAP offers novel and complementary information as compared to SNP-based analysis approaches and is straightforward to implement with existing open-source statistical software tools. Serum lipid levels are established determinants of cardiovascular disease morbidity with well-described heritability. Indeed, meta-analysis of data arising from recent genome-wide association studies has identified common genetic variants in at least 95 loci associated with low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglycerides (TG) and total cholesterol (TC) Mixed modeling of Meta-Analysis P-values (MixMAP), draws strength from available information across all SNPs within a pre-defined region without requiring first-stage data reduction. That is, it is not necessary to eliminate redundancy among SNPs in high linkage disequilibrium (LD) within a locus. By incorporating all available information about SNPs within a locus, MixMAP results in increased sensitivity for identifying loci with multiple SNPs of moderate significance, as evidenced in the applications described below.In order to address this, we apply a mixed effects modeling paradigm that uses SNP-level meta-analysis p-values to arrive at formal analytic characterization of underlying locus-level effects on complex disease phenotypes. Through application of principled, well-vetted statistical concepts for valid and reliable inference, this approach, termed et al.et al. framework is based on fixed effects parameters (representing pathway effects) while MixMAP involves generating prediction intervals for latent (locus-level) effects.Applications of hierarchical linear and generalized linear models for analysis of data arising from genetic association studies have been described previously. Specifically, use of mixed effects models for family studies is common to account for correlation arising from familial level clustering where a random effect for family is included in the model (see for example http://cran.r-project.org/ and http://people.umass.edu/foulkes/software.html.).The purpose of this manuscript is to describe the MixMAP algorithm and to illustrate its application under a range of conditions. Application of MixMAP to two independent datasets of SNP associations with LDL-C, a causal risk factor for atherosclerotic cardiovascular disease (CVD), reveals that MixMAP offers novel and complementary information as compared to traditional analysis approaches. Notably, as an analytic tool that selects loci with multiple moderate to strong signals, and one that uses single SNP-level summary data  and the Penn Coronary Artery Calcification (PennCAC). These revealed that application of MixMAP: (1) supports published LDL-C loci; (2) suggests novel LD-CL genes and (3) complements a single-SNP testing approach, as described in detail below. Additionally, the results of simulation studies designed to characterize the performance of MixMAP under a range of conditions, also described below, support the concept that MixMAP serves as a complementary analysis strategy. To begin, we briefly outline the MixMAP algorithm and the available GLGC and PennCAC data. Further details are provided in the Material and Methods.MixMAP is a statistical framework that uses the results of single SNP-level analysis to test formally for locus-level association. The MixMAP approach is designed to identify loci involving multiple SNPs each with moderate effects on the trait that may not be detected by single SNP analysis. Loci that are detected by a single SNP approach may also be detected by MixMAP if multiple SNPs have modest association across the locus. The primary inputs to the MixMAP algorithm are: (a) p-values corresponding to single SNP tests of association with a trait (e.g. LDL-C); (b) a mapping of SNPs to genomic regions. Additional SNP and gene-level covariate information, such as gene size, number of SNPs per Kb, number of recombinant hotspots per Kb and average linkage disequilibrium can be incorporated.Step 1:] Fit a mixed effects model to inverse normally transformed (ranked) Step 2:] Predict random locus-specific effects using empirical Bayes estimation; [Step 3:] Calculate corresponding prediction intervals using a Bonferroni corrected threshold; and [Step 4:] Report a locus as statistically meaningful if the upper limit of the prediction interval corresponding to the locus effect is less than The MixMAP algorithm is summarized as follows, with additional detail provided below: [http://www.broadinstitute.org/mpg/pubs/lipids2010/); and (2) SNP level The current applied investigation focuses on loci for LDL-C, an important causal factor for CVD. Data arising from two independent data-sets are considered: (1) reported and publicly available meta-analysis SNP level A total of 31827 SNPs in 2960 genes that are common to both GLGC and PennCAC are used in the current investigation. As an illustrative example, we chose to focus on the set of SNPs included on the IBC array which was specifically designed to provide SNP coverage in putative candidate CVD genes as well as emerging loci at the time of design http://www.broadinstitute.org/mpg/snap/)  additional loci are supported by MixMAP that are not detected using the single SNP signal threshold in the GLGC data . While tHere we apply MixMAP to a small study (PennCAC) that generated IBC array SNP data. In terms of independent support, we treat published GLGC data as the gold standard to which PennCAC is compared. This analysis includes 31585  in 2944 (of 2960) genes that remain after filtering out those with MAFIn PennCAC, an additional hold of  . None ofTPR), defined as the proportion of true signal genes that are correctly identified; the false discovery rate (FDR), given by the proportion of selected genes that are not associated with the trait; and the false positive rate (FPR), defined as the proportion of truly uninformative genes that are incorrectly selected as significant. Explicit definitions of TPR, FDR and FPR are given in In order to evaluate the performance of MixMAP relative to single SNP analysis, we investigate the ability to detect informative loci and the likelihood of false findings as functions of gene level effect size (measured by the shift parameter in a two-component Gaussian mixture distribution), the number of informative genes  and coverage . Details of the simulation approach are described in Materials and Methods below. Simulation results are reported in terms of: the true positive rate . Third, although many loci for LDL-C have been identified through association studies at In GLGC data, MixMAP confirmed association for over A more detailed description of the 12 genes/loci detected by MixMAP alone is provided in Supplementary Materials. For many of these 12 loci  the GLGC single SNP test p-value provided suggestive evidence of association  using LDL-C as an example in part because the large GLGC dataset for LDL-C provides an external reference for any MixMAP findings. In PennCAC, no individual SNPs meet criteria for association with LDL-C using the conservative genome-wide Bonferroni correction . This is related in expectation to Based on this model formulation, the best linear unbiased predictor of the random effect for gene In general, we are interested in testing the null hypothesis of no association between a given gene and the trait. To this aim, we construct a one-sided prediction interval for the true gene-level effect, given by For all simulation studies, random gene level effects, Supporting Information S1Clinical Studies.(PDF)Click here for additional data file.Supporting Information S2Summary of suggested novel genes/loci identified in GLGC data by MixMAP.(PDF)Click here for additional data file.Table S1Empirical Bayes (EB) estimates and corresponding prediction intervals for MixMAP supported genes in GLGC. The Empirical Bayes estimate and corresponding prediction variance for each gene are used in the construction of an associated one-sided prediction interval. 1The upper limit of this interval is reported using a Bonferroni corrected α = 0.05/(2960). An upper limit that is less than 0 implies the gene's random effect  is significantly less than 0. Since negative transformed p-values that are large in absolute value correspond to small p-values on the original scale, this implies the gene as a whole has a significant effect on LDL-C. *Highlighted rows correspond to novel loci not identified using single SNP analysis.(PDF)Click here for additional data file.Table S2Empirical Bayes (EB) estimates and corresponding prediction intervals for MixMAP supported genes in PennCAC. The Empirical Bayes estimate and corresponding prediction variance for each gene are used in the construction of an associated one-sided prediction interval. 1The upper limit of this interval is reported using a Bonferroni corrected α = 0.05/(2944). An upper limit that is less than 0 implies the gene's random effect  is significantly less than 0. Since negative transformed p-values that are large in absolute value correspond to small p-values on the original scale, this implies the gene as a whole has a significant effect on LDL-C. None of these loci were identified using single SNP analysis.(PDF)Click here for additional data file.


Riboswitches are RNAs that modulate gene expression by ligand-induced conformational changes. However, the way in which sequence dictates alternative folding pathways of gene regulation remains unclear. In this study, we compute energy landscapes, which describe the accessible secondary structures for a range of sequence lengths, to analyze the transcriptional process as a given sequence elongates to full length. In line with experimental evidence, we find that most riboswitch landscapes can be characterized by three broad classes as a function of sequence length in terms of the distribution and barrier type of the conformational clusters: low-barrier landscape with an ensemble of different conformations in equilibrium before encountering a substrate; barrier-free landscape in which a direct, dominant “downhill” pathway to the minimum free energy structure is apparent; and a barrier-dominated landscape with two isolated conformational states, each associated with a different biological function. Sharing concepts with the “new view” of protein folding energy landscapes, we term the three sequence ranges above as the sensing, downhill folding, and functional windows, respectively. We find that these energy landscape patterns are conserved in various riboswitch classes, though the order of the windows may vary. In fact, the order of the three windows suggests either kinetic or thermodynamic control of ligand binding. These findings help understand riboswitch structure/function relationships and open new avenues to riboswitch design. Riboswitches are RNAs that modulate gene expression by ligand-induced conformational changes. However, the way that sequence dictates alternative folding pathways of gene regulation remains unclear. In this study, we mimic transcription by computing energy landscapes which describe accessible secondary structures for a range of sequence lengths. Consistent with experimental evidence, we find that most riboswitch landscapes can be characterized by three broad classes as a function of sequence length in terms of the distribution and barrier type of the conformational clusters: Low-barrier landscape with an ensemble of conformations in equilibrium before encountering a substrate; barrier-free landscape with a dominant “downhill” pathway to the minimum free energy structure; and barrier-dominated landscape with two isolated conformational states with different functions. Sharing concepts with the “new view” of protein folding energy landscapes, we term the three sequence ranges above as the sensing, downhill folding, and functional windows, respectively. We find that these energy landscape patterns are conserved between riboswitch classes, though the order of the windows may vary. In fact, the order of the three windows suggests either kinetic or thermodynamic control of ligand binding. These findings help understand riboswitch structure/function relationships and open new avenues to riboswitch design. Riboswitches are RNAs in the untranslated (UTR) regions of messenger RNAs (mRNAs) that can undergo a structural transition in response to a highly specific intracellular ligand Riboswitches are composed of two major RNA domains: an aptamer domain, which binds the ligand, and an expression platform, which controls gene expression . The aptOne of the most common forms of gene control by the expression platform is the transcription terminator hairpin. As illustrated in Another example of gene control by the expression platform is the folding of the Shine-Dalgarno sequence. The Shine-Dalgarno sequence is a section of the expression platform and the ribosome binding site in prokaryotes. In the presence of ligand, the Shine-Dalgarno sequence forms a double-stranded RNA (anti-SD), which prevents the ribosome from binding and precludes translation of the gene. Transcription termination and Shine-Dalgarno sequence sequestration are both mechanisms that riboswitches use to control gene expression; however, they accomplish this by acting on two different processes within the cell in vivo, the RNA folding process must be investigated. RNAs begin to fold as they are transcribed in the cell and are efficiently directed toward a stable conformation through fast base-pairing interactions (∼100 ms) in vivoTo understand riboswitch gene control in vivo folding places limits on the time permitted for RNA-ligand equilibration. First, in the absence of transcriptional pause sites, RNA polymerase transcribes nucleotides quickly; ligand binding occurs before the polymerase reaches the end of the expression platform (in vivo and that these concentrations surpass the in vitro dissociation constant (KD) D of the aptamer-ligand complex is generally near the cellular concentration of the ligand Recently, studies have elucidated two mechanisms of ligand binding in riboswitches: thermodynamic and kinetic platform , bottom.pbuE riboswitch in vivo. For example, although both pbuE and add riboswitches bind adenine, pbuE demonstrates kinetic control, while add shows thermodynamic control A riboswitch may use both strategies, as shown for the Riboswitch folding is a multi-step hierarchal process, involving interactions between base-pairs , base stacking, hydrogen bonding, and tertiary interactions between distant or proximal nucleotides. While gene control is affected by changing secondary structure, local changes also occur to adopt a binding pocket specific for a small ligand. Modeling RNA interactions on the global and local levels is thus required to fully grasp the switching process . It is believed that most of the structural scaffolding, which includes secondary and tertiary interactions, is quickly formed, while the addition of the ligand only causes specific tertiary contacts. For example, Stoddard et. al. 1In cooperation with the pre-organized aptamer, key tertiary interactions form when the ligand binds, and prior simulations have also shown how this response to the ligand occurs. An atomic-level computer simulation of the S-adenosylmethionine (SAM) aptamer To better interpret the folding process of RNA, we use the perspective of the “new view” of protein folding, which relies on the concept of a free energy landscape In general, biological molecules take advantage of a funnel-shaped landscape representing many high-energy (denatured) conformations and few low-energy states. This arrangement permits the sequence to search the astronomical number of conformations directly and efficiently. In a “smooth” energy landscape, there are few low-energy structures in the lowest energy portion of the funnel, whereas a “rough” energy landscape has more low-energy structures with barriers between them. In the latter, each of the low-energy structures has a smaller funnel leading to it. If the landscape is smooth and has a single minimum, the minimum free energy occurs near the native state. This situation is called “downhill folding tenA TPP riboswitch. The nature of the unbound state, the change in secondary structure, and the effects of the expression platform on folding are all questions we address here by deriving a novel energy landscape model and validating our predictions with experimental measurements. Studies on the full riboswitch, aptamer and expression platform, are still lacking. Here, we simulate in vivo formation of structures by calculating the energy landscape of secondary structures sequentially from short to full length sequence, without any ligand, at 1 nt increments. Prediction of individual RNA secondary structures at different lengths is performed with a set of programs from the Vienna RNA folding package We use this procedure to analyze ten riboswitches from seven different classes, by the technique we developed in sensing window encompasses the lengths at which the riboswitch adapts to different structures, including the ligand-competent form. Overall, the ligand-competent and non-ligand-competent structures are inherent to the energy landscape  or anti-SD . In this time frame, the energy landscapes describe a spontaneous isomerization to the stable antiterminator/anti-SD form. We propose that this window decides the ultimate fate of the riboswitch: If the RNA is ligand-bound, it does not isomerize to antiterminator/anti-SD form, and without ligand, the RNA forms the thermodynamically-favored antiterminator/anti-SD form. In the functional window, the final set of nucleotides of the expression platform form the terminator hairpin/antiterminator or sequester/open ribosome binding site, which are energetically favored. ligands . At the tenA and thiM thiamine pyrophosphate (TPP) riboswitches, each energy landscape window correlates with several interesting experimental properties. First, the RNA favors the ligand-competent form in the sensing window  in vivo gene regulation is greater than the binding affinity found in vitro (KD).Overall, this order of windows is characteristic of kinetic control, where the choice of folding pathway occurs early in transcription. High concentrations of ligand both stabilize the ligand-competent aptamer soon after it is transcribed and exclude non-ligand-competent forms Candidatus Desulforudis audaxviator belongs to a novel class of riboswitches found to bind the second messenger cyclic di-guanosine monophosphate tenA and thiM, the sensing window contains ligand-competent and non-ligand-competent structures together on the energy landscape, separated by a small energy barrier . The folding pathway starts with a sensing window, where ligand-competent and non-ligand-competent structures are in equilibrium. The downhill folding window that follows shows a tendency to isomerize to the anti-SD structure. Finally, in the functional window, the anti-SD forms a separate cluster from functionally-opposing structures, which have open Shine-Dalgarno sequences. This functional window does not display a clear separation of clusters as in thiM, though it has a high energy barrier between sets of conformations . Experimental studies on kinetic or thermodynamic control of ligand binding are not yet available, though our predicted structures contain all conserved features of the ligand-bound structure (The molybdenum-cofactor binding tructure .metI leader from Bacillus subtilis binds S-adenosylmethionine (SAM) and exhibits dramatic gene silencing in the presence of ligand (∼12%→75% termination in presence of ligand) The mgtE riboswitch from Bacillus subtilismgtE ligand binding because the sensing window occurs during sequence-lengths shorter than full length. No kinetic studies have yet been performed on this riboswitch to the best of our knowledge.The pbuE riboswitch alters its structure in response to adenine only at short lengths pbuE favors an adenine-binding-competent fold at short lengths, in the sensing window, where loops L2 and L3 and stem P1 forms pbuE riboswitch differs from all other classes, because the ligand-competent structure is not the mfe at any point in the windows. In strong agreement with optical trapping assays of the pbuE aptamer domain The pbuE is followed by a functional window, in which two pathways become possible. Both Set 1 and 2 structures favor terminator hairpins  (The sensing window in uctures) . Ligand-pbuE RNA highly favors non-ligand-bound states as indicated by a downhill folding window toward the non-ligand-competent mfe. Adenine-competent structures exist on the landscape, but are much higher in energy. This suggests that the ligand must stabilize the RNA to prevent isomerization to more energetically favorable non-ligand-competent structures. This behavior agrees with experimental studies pbuE riboswitch is not responsive to ligand, meaning that the RNA does not fold into a ligand-competent structure when adenine is subsequently added to solution.The energy landscape for the full length pbuE suggests kinetic control. This finding is also in agreement with experimental results Since the sensing window occurs early in transcription, xpt-pbuX guanine-sensing riboswitch has a similar structure and sequence to pbuE, specific nucleotides in its aptamer domain bind guanine. Once the xpt aptamer domain is transcribed, it forms the ligand-competent structure  were given to the riboswitch to fold into the preferred structure in the downhill folding window, the terminator would always form and gene regulation would not depend on the presence or absence of guanine. Whichever structures are formed at the end of the functional window (ligand-competent/terminator or non-ligand-competent/antiterminator) are thus kinetically trapped through the downhill folding window. Gene regulation can thus follow.For gene regulation to occur, we propose a model of kinetic control. The structures in the sensing window likely exchange at equilibrium, much like in add selectively binds adenine add riboswitch operates on the basis of thermodynamic control of adenine. Therefore, we propose that the following sequence of energy landscape windows is the hallmark of thermodynamic control . The authors propose that the riboswitch operates by kinetic control. Pausing, however, may provide ample time for the full length riboswitch to bind the ligand, alter its structure, thereby allowing thermodynamic control.The preQ1 riboswitch is the smallest riboswitch class known and requires a pseudoknot interaction in the aptamer domain for ligand binding Our results show that a downhill folding window occurs from positions 42 to 53, where the mfe is a non-ligand-competent structure . Higher-We have developed a computational approach based on energy landscapes that describes riboswitch conformational ensembles at different sequence ranges as the riboswitch elongates to full length and analyzed the resulting landscapes for ten riboswitches. Our analysis suggests variations in riboswitch control of gene regulation and patterns that describe either kinetic or thermodynamic control.In the case of kinetic control of riboswitches, base-pairing occurs within milliseconds as the RNA emerges from the RNA polymerase and the ligand-competent aptamer structure emerges in the sensing window. This ligand-competent structure is in equilibrium with other non-ligand-competent structures. Exchange between stable states occurs up until the downhill folding window, with either bound or unbound ligand, and the final RNA structure remains stable during the functional window.In the alternative thermodynamic control, structural changes can be induced by the ligand throughout the elongation process. Other studies by Nudler et al. Because all RNA folding algorithms are imperfect, and sampling of suboptimal structures at elevated temperatures entails additional approximations, the results presented here are subject to these standard uncertainties. We also cannot account for all biologically relevant events, such as transcription pause sites pbuE aptamer Support for switching states comes from our energy landscape analysis, where shorter-than-full length riboswitch transcripts form different low-energy structures in the sensing window. The low energy barrier and natural tendency for fast RNA base-pairing implies rapid structural interconversions at ambient temperatures. Indeed, thermal excitation for overcoming an energy barrier of 5–10 kcal/mol has been postulated for the pbuE and preQ1) examined here. Even for these riboswitches, ligand-competent structures exist within the cluster of accessible conformations though. The centroid of a cluster may be closer to the reference standard RNA structure Another remarkable property of the sensing window is that the ligand-bound conformation is the minimum energy structure on the energy landscape. As a result of the aptamer's upstream location on the riboswitch construct, the ligand-competent state forms as soon as it emerges from the polymerase. Some studies suggest that ligand-competent states do not occur spontaneously in the majority of riboswitches E. coli phage MS2 RNA, which requires rearrangement in order to restrict ribosome binding and control gene expression . This property exists in other structurally rearranging RNAs, such as the pression  [85]. Thpression . These ein vivo. Our computational results support this and broaden this notion to some translationally-acting riboswitches as well (thiM and moaA). If the ligand binding regime of the sensing window does not occur at full length, then the riboswitch cannot bind ligand reversibly and undergo the requisite structural modulation.An essential function of riboswitches is their ability to transmit ligand binding into gene regulation. Kinetically-controlled riboswitches perform binding and structural rearrangement during transcription, while thermodynamically-controlled riboswitches display structural reversibility in response to ligand post transcription in vitro methods. To further explore further our 2D models, we also perform illustrative 3D folding on two riboswitch aptamers . Free energy of folding for all structures generated is then re-calibrated at 37°C using RNAeval to be physiologically relevant and for purposes of comparison. To attain a representative view of the energy landscape through time, we iteratively sample and measure the energy landscape from 64% of the full length of RNA, in increments of one nucleotide. Prior to 60% of the full length RNA, we found that most riboswitches are composed solely of the RNA aptamer domain, and thus would not reflect the dual-structure nature of the landscape through time.Suboptimal structure sampling Structure comparison was performed using the base pair distance in RNAdistance Rcluster package. We used Partitioning Around Medioids (PAM), a partition clustering algorithm, specifying the number of clusters to be two (k = 2). This method is highly effective and amenable to our methods as it predicts clusters by minimizing a sum of dissimilarities, which we deal with implicitly as the base pair distance. For quantifying the strength of clustering and as a surrogate quantity for the presence of two clusters, the average silhouette width was used with a threshold of ≥0.4. The average silhouette coefficient for all points (SC) ranges from 0 to 1, indicating a poor or well-clustered result respectively. For a full description, see ref Free energy of folding versus base pair distance is used as an approximate measure of the energy landscape. Once the landscape is generated, clustering is performed in the statistical software We generate energy landscapes for all riboswitch sequence lengths from 64% of full length until the end of the expression platform. We then analyze the landscape for the presence of clusters and particular shapes. Barrier-limited landscapes are defined by the following: (1) alternative, low-energy structures within 2.0 kcal/mol of each other (2) an energy barrier between 5 and 10 kcal/mol between the two states (3) a funnel-type topology within the vicinity of the local minima. Using the criteria above, a “sensing window” is defined by at least 5 consecutive nucleotide lengths that meet the definition of “barrier-limited.” We also found that, within the same RNA construct, the energy landscape may differ. Those landscapes that did not fit the criteria of barrier-limited landscape and had a barrier of <5 kcal/mol are defined as “funnel” and if the barrier is >10 kcal/mol defined as “cluster.” If five or more consecutive nucleotides display similar funnel landscapes, it is a “downhill folding window.” Conversely, if five or more consecutive nucleotides display cluster landscapes and the functionally-opposing structures occupy the sets, it is a “functional window.Figure S1Comparison between computationally predicted and experimentally verified structures. Comparison between computationally predicted and experimentally verified structures. Ligand binding domains of experimentally validated structures are shown. Nucleotides which differ between the two structures are marked in blue (predicted structure is base-paired), red (predicted structure is single-stranded), orange , and green (agreement). Dashed lines represent base pairs present in the computationally predicted structure.(EPS)Click here for additional data file.Figure S2Predicted tertiary structures of the SAM and thiM TPP aptamer. Predicted tertiary structures of the SAM and thiM TPP aptamer. (a) Overlap of the predicted and wild-type SAM aptamer of Thermoanaerobacter tengcongensis in the ligand-free form. (b) RMSD trajectory  over the course of the 3D folding. (c) Overlap of the ligand-free predicted and ligand-bound wild-type TPP aptamer of Escherichia coli. (d) RMSD trajectory  over the course of the 3D folding.(EPS)Click here for additional data file.Figure S3Folding pathway for the E.coli phage MS2 RNA. Initially, two structures are possible which the RNA may fold into (86–117 nt). The RNA was found to isomerize into the bottom structure. The energy landscape from 122–136 nt is downhill folding and accommodates such isomerization to this specific mfe structure.(EPS)Click here for additional data file.Figure S4The folding pathway of the synthetic MDV1 RNA. The following is an explanation from the original article by Kramer and Mills:Both hairpin B and hairpin T are present during chain growth but cannot coexist on the same molecule. Hairpin B is initially favored during chain growth, prior to formation of hairpin T. Once MDV1 grows sufficiently long (61–71 nt), hairpin T is more stable than hairpin B. Additional chain growth leads to the dissociation of hairpin T in favor of the formation of hairpin C and the reformation of hairpin B, which together contributes more to the stability of the RNA than hairpin T alone.  The energy landscape from 76–96 nt favors downhill folding and thus isomerization to the structure containing hairpin B and hairpin C.(EPS)Click here for additional data file.Figure S5Clusters in the energy landscape as a function of sequence length. Charts display the number of clusters in the energy landscape at 1 nt resolution. Significant clusters were defined by a silhouette coefficient ≥0.4 (see ≥0.4 see .(EPS)Click here for additional data file.Table S1Tertiary contacts used as input into NAST.(DOC)Click here for additional data file.Text S1Potential 3D structures of aptamers arise as an extension of 2D predictions.(DOC)Click here for additional data file.Dataset S1Wild-type riboswitch sequences used in this study.(DOC)Click here for additional data file.


Angiotensin-converting enzyme (ACE) regulates normal blood pressure and fluid homeostasis through its action in the renin-angiotensin-system (RAS). Ace-/- mice are smaller in size, have low blood pressure and defective kidney structure and functions. All of these defects are cured by transgenic expression of somatic ACE (sACE) in vascular endothelial cells of Ace-/- mice. sACE is expressed on the surface of vascular endothelial cells and undergoes a natural cleavage secretion process to generate a soluble form in the body fluids. Both the tissue-bound and the soluble forms of ACE are enzymatically active, and generate the vasoactive octapeptide Angiotensin II (Ang II) with equal efficiency. To assess the relative physiological roles of the secreted and the cell-bound forms of ACE, we expressed, in the vascular endothelial cells of Ace-/- mice, the ectodomain of sACE, which corresponded to only the secreted form of ACE. Our results demonstrated that the secreted form of ACE could normalize kidney functions and RAS integrity, growth and development of Ace-/- mice, but not their blood pressure. This study clearly demonstrates that the secreted form of ACE cannot replace the tissue-bound ACE for maintaining normal blood pressure; a suitable balance between the tissue-bound and the soluble forms of ACE is essential for maintaining all physiological functions of ACE. Both isoforms are synthesized from a single gene using tissue specific promoters and have distinct and tissue-specific functions in the body The Renin-Angiotensin System (RAS) is a coordinated hormonal cascade that modulates fluid and electrolyte balance as well as blood pressure regulation Studies with ACE knockout mice revealed additional roles of ACE beyond blood pressure regulation, especially in kidney functions, development and male fertility ACE is expressed on the cell surface as type I ectoprotein, with a long ectodomain containing the enzymatic active site, short cytoplasmic domain and a transmembrane domain To investigate the relative roles of secreted and cell-bound ACE in various physiological functions of ACE, we have generated a transgenic mouse model which expressed only the secreted form of sACE, without any cell-bound form. This mutant ACE protein lacks the transmembrane and the cytoplasmic domains and, therefore, is constitutively secreted in the circulation. Our results demonstrated that the secreted ACE could not restore normal blood pressure of Ace-/- mice, although their kidney functions were normal. This study demonstrates that a cell-bound ACE is critical for maintaining normal blood pressure; a secreted ACE cannot substitute for the cell-bound form for this function.We used Wt rabbit sACE cDNA to generate esACE, by deletion of the transmembrane and cytoplasmic domain coding region. The esACE cDNA was then used to generate Tie-esACE construct, as described previously The tie-esACE transgene was microinjected into the pronuclei of FVB strain zygotes using standard techniques. Adult FVB tie-esACE-BGHpA (TeS) transgenic founder mice  were mated with Ace+/− FVB mice to generate Ace+/− TeS+/− mice. Interbreeding between male and female Ace+/−, TeS+/− mice within the same line was performed to generate the Ace-/-, TeS/TeS (TeS). Genotyping of all mice was performed by Southern Blotting as described below. Expression of esACE in the transgenic line was confirmed by Western Blotting of the serum using anti-ACE antibody. All of the mice described in this study, including Wt, KO, Tie-esACE (TeS) and Tie-sACE (Ts), were of the FVB strain . All mouAce-/- Ts/Ts (Ts) control mice were generated by mating Ace+/− Ts/Ts males with Ace-/- Ts/Ts females as described previously Ace gene value in the same genomic DNA sample using Imagequant software. The endogenous Ace genotype is determined by the presence of a wild-type 6.6 kB SacI genomic fragment or the disrupted 8.4 kB SacI (Ace-/-) genomic fragment Southern blot genotyping was performed as described previously utilizing Sac I digestion of genomic tail snip DNA The enzymatic activity of ACE was assayed using Hip-His-Leu as a substrate and measuring fluorimetrically the His-Leu liberated at 5 mM of Hip-His-Leu. Serum (1 µl) from retro-orbital eye bleed or tissue homogenates of the transgenic mice were used to measure ACE enzymatic activity. Activity values are reported as µmoles His-Leu per per µl of serum or 25 µg of tissue protein extract liberated from Hip-His-Leu after one-hour incubation at 37°C.Blood was collected by retro-orbital sinus plexus eye bleed under brief isoflurane anesthesia and plasma renin activity was measured as described before Serum creatinine levels were determined by previously described alkaline picrate method For each genotype, blood from four age matched adult mice was pooled to achieve a 1 ml plasma sample. Samples were concentrated on C18 columns , evaporated to dryness, and reconstituted in 0.9% NaCl, 0.03% acetic acid and 0.1% BSA). The levels of Ang II were quantified by RIA ad libitum. The data indicates average daily (24 h) water consumption and urine volume produced for five consecutive days for each of five mice of the same genotype. Urine osmolality was measured for each mouse using the Osmette A  freezing point osmometer according to the manufacturer's instructions. Triplicate readings were performed on the urine collected as indicated above.Age-matched adult mice  were individually placed in a Nalgene metabolic cage supplied with powdered standard chow and water Blood pressure was measured by two independent methods, by radiotelemetry and tail-cuff plethysmography. For telemetric measurements, the mice were anesthetized with isoflurane (3% in an oxygen stream). The BP transmitter  was implanted as follows: the catheter was inserted into the left common carotid artery, and the transmitter was positioned in the right flank Kidneys from age-matched adult mice were paraffin embedded, cross-sectioned at 2.5 µm thickness and hematoxylin and eosin stained by the Histology Core . Immunohistochemistry was performed following de-paraffinization as described previously Kidneys from Ace+/− or TeS mice were homogenized in 20 mM Tris HCl, 150 mM NaCl, 0.1% Triton X-100, protease inhibitors and phosphatase inhibitors leaving on ice for 30 min and spun at 14000 rpm at 4°C. The homogenates  were separated on 12% SDS-PAGE and Western blotted by anti-renin antibody . The loading control was confirmed by Western blotting against actin.t test, comparing experimental mice to the Wt mice or the KO mice as indicated in the figure legend. The statistical significance has been indicated in the figure legends.Data are presented as arithmetic means and variations as 95% confidence interval of the mean. Significance values were obtained by unpaired in vitro by transiently expressing in human cells (HT1080). The esACE protein was expressed in these cells with similar expression as the sACE, with the expected size. This cellular form of esACE is the underglycosylated precursor form of ACE which is not transported to the cell surface; as indicated by two forms (the precursor and the glycosylated cell surface expressing form) for the sACE  Since esACE was exclusively secreted into the blood flow from vascular endothelial cells, no significant ACE staining was observed in the kidney section of TeS mice by immunohistochemistry . The AceAce-/- mice exhibit hypoplasia of renal cortex; in the renal cross section, the area outside the dotted line is markedly reduced compared to the Ace+/+ mice . The devTo address whether the restored kidney structures correspond to normal kidney functions, we compared their urine concentrating ability by measuring water intake, urine output and urine osmolarity. The overall fluid homeostasis was measured by the amount of urine output and water uptake over a period of five days. Consistent with previous findings, the Ace-/- mice exhibited significantly high urine output as well as high water uptake, presumably due to the defect in concentrating urine . HoweverWe tested the integrity of local RAS by measuring kidney-associated renin and Ang II levels of TeS mice. Renin, a critical regulator of local RAS, was analyzed in kidney homogenates. The TeS mice expressed similar levels of kidney-associated renin as compared to Ace+/− mice . To furtTo examine whether restored renal phenotype, normal levels of circulating ACE, renal and plasma Ang II, plasma renin activity are sufficient to maintain the normal blood pressure of TeS mice, we conducted two independent experiments, computerized tail-cuff plethysmography and radiotelemetry, to measure the systolic and diastolic blood pressures of the mice. The TeS mice exhibited low systolic blood pressure, comparable to that of Ace-/- mice, whereas Wt (Ace+/+) or Ace+/− mice both showed normal blood pressure . The sysIn vitro studies using both sACE and gACE proteins show that deletion of cytoplasmic domain leads to elevated levels of soluble ACE Ectodomain shedding of ACE generates an enzymatically active secreted form, whose function is currently unknown. We hypothesized that a suitable balance between the cell-bound and secreted forms of ACE is critical for maintaining the normal physiological functions of the enzyme. To test this hypothesis, we generated transgenic mice expressing a secreted sACE, in the absence of any cell-bound ACE. Our results showed that secreted sACE could not maintain normal blood pressure; however, it was sufficient for restoring normal growth, development, kidney structures and functions, of Ace-/- mice. This strongly indicates the requirement of endothelial cell-bound sACE for maintaining normal blood pressure. This conclusion is supported by our previous results with another transgenic mouse, in which sACE was restrictedly expressed in the kidney proximal tubular cells (Gs mice) Inhibition of ACE enzymatic activity by selective ACE inhibitors blocks the generation of Ang II and thereby acts as a suitable approach for the treatment of high blood pressure. However, it remains unclear whether the Ang II generating function of ACE is the only link between this protein and the maintenance of normal blood pressure. Our study indicates that the level of Ang II is not an accurate indicator of ACE function in blood pressure maintenance. Although, both the TeS mice and the control Ace+/− mice expressed similar levels of renal and plasma Ang II , 6B, theSo, why is cell-bound ACE essential for blood pressure maintenance? The concept of local vs. systemic RAS may be relevant to this question. The components of the RAS exist in many organs and are postulated to regulate tissue specific Ang II production


Neurogenesis in the subgranular zone of the mammalian hippocampal dentate gyrus contributes significantly to brain neuroplasticity. There is evidence that inflammation of the central nervous system inhibits neurogenesis but peripheral inflammation such as antigen-induced arthritis may rather enhance neurogenesis. Manifest arthritis is associated with symptoms such as pain and altered locomotion indicating that peripheral inflammation is associated with changes of both the immune system and the nervous system. This raises the intriguing question whether immune or neuronal factors or both actually drive changes of neurogenesis. Here we explored hippocampal neurogenesis in the rat during chronic antigen-induced arthritis in the knee joint. We analyzed neurogenesis in control rats, and in rats which were immunized for the antigen producing arthritis but which did not show arthritis and neurological symptoms, and in rats in which antigen injection into the knee produced manifest local inflammation and symptoms such as pain at the inflamed knee and altered locomotor behavior. Neurogenesis was assessed by quantifying bromodeoxyuridine-positive cells in sections of the complete hippocampal dentate gyrus. Compared to control animals, rats with antigen-induced arthritis presenting manifest local inflammation, hyperalgesia at the inflamed knee and significantly altered locomotion exhibited a significant increase of bromodeoxyuridine-positive cells. However, a similar increase in the number of such cells was found in rats which were only immunized against the antigen, but in which no local inflammatory response was induced and which thereby neither showed hyperalgesia nor alterations of locomotion. Thus we conclude that in peripheral immune-mediated arthritis the activation of the immune system in the process of immunization is the causal factor driving enhanced neurogenesis, and neither the local enhancement of inflammation nor the activation of the nervous system leading to neurological symptoms such as pain and altered locomotion. It seems noteworthy to further explore the clinical importance of this neuroimmune interaction. Peripheral inflammation such as arthritis causes pain, guarding behavior, and other pain-related disturbances such as fatigue Neurogenesis in the subgranular zone (SGZ) of mammalian hippocampal dentate gyrus (DG) is an important mechanism of brain neuroplasticity. Adult neurogenesis whereat neural stem/progenitor cells (NSCs) proliferate into neuronal or glial progenitors The dentate gyrus also responds to different types of pathophysiology with significant changes in neurogenesis. Adult neurogenesis may increase in the context of acute pathophysiological insults but this does not necessarily represent beneficial adaptations of the hippocampal network which promote reorganization and recovery. In the context of epilepsy and stroke, significant portions of newborn neurons form aberrant dendritic arborization and connectivity which impair hippocampal function AIA is a suitable model of immune-mediated arthritis such as human rheumatoid arthritis because it shows typical features of rheumatoid arthritis such as a progression from an acute inflammatory to a chronic inflammatory/destructive state with flare-up reactions This study was carried out in strict accordance with EC regulations for the care and use of laboratory animals. The protocol was approved by the Thuringian state authorities for animal protection (registration number 02-013/08). All injections for immunization and BrdU application, and induction of monoarticular AIA were performed under short anesthesia with 3% isoflurane.ad libitum. AIA was induced as reported previously Mycobacterium tuberculosis strain H37RA  were injected subcutaneously twice with a one-week interval between immunizations  were used for the studies. Lewis rats are particularly susceptible to AIA izations . After aAnimals were divided randomly into 5 experimental groups. In 7 rats AIA was induced (AIA group). Another group of 10 animals were only immunized but did not get the intraarticular injection of mBSA [“immunized control” (IC) group]. Another 7 rats received no treatment [“naïve control” (NC) group], 7 rats underwent only X-ray exposure (CXray group), and 3 rats were only immunized but they were neither exposed to X-ray nor were they tested for changes in pain and locomotor behavior (IC-wt = “immunized control without testing” group). The IC-wt group consists of only 3 animals, because of tissue-damage after brain removal.Primary hyperalgesia at the inflamed knee was assessed with a dynamometer  Weight bearing as a functional measure of pain-related guarding behavior of the inflamed hindlimb was assessed with an incapacitance tester . For that purpose animals were placed with both hind paws on scales inside a plastic cage. The weight force resting on the two scales was consecutively measured for three times after animals acclimated and sat calm in the cage. From these averaged values, the relative weight (in %) resting on the inflamed hindlimb was evaluated [weight on inflamed hindlimb × 100%/(weight on inflamed hindlimb+weight on non-inflamed hindlimb)] as described previously Videoradiographic analysis was performed as reported in detail previously In order to identify proliferating cells all animals received daily intraperitoneal injections of bromodeoxyuridine  on 5 consecutive days beginning on day 1 of the experimental time-course under short anesthesia with 3% isoflurane. On day 42, animals were deeply anesthetized with 120 mg/kg sodium thiopentone injected intraperitoneally  and transcardially perfused with 4% ice-cold phosphate-buffered paraformaldehyde (PFA) see . Whole b2O2 (30 min at room temperature), rinsed in TBS , treated with 2 N HCl for DNA denaturation (30 min at 37°C) and rinsed in 0.1 M boric acid (10 min at room temperature), then rinsed in TBS , incubated in blocking solution  followed by incubation with the primary antibody  in blocking solution at 4°C overnight. After rinsing in TBS , sections were incubated in blocking solution (30 min at room temperature) and incubated with the secondary antibody  for 2 hours at room temperature. After rinsing in TBS  the slices were incubated in ABC solution  for 1 hour at room temperature. After rinsing in TBS  the slices were processed using diaminobenzidine hydrochloride  as the chromogen, rinsed in TBS  and finally mounted onto gelatine-coated slides, air-dried and coverslipped with Entellan .For immunoperoxidase staining, free-floating sections were rinsed in TBS , incubated in 0.6% H2O2-treatment. Sections were incubated with primary antibodies , 1∶500, Chemicon, Temecula, CA, USA) in blocking solution at 4°C overnight. After rinsing in TBS , sections were incubated in blocking solution (30 min at room temperature) and incubated for 2 hours at room temperature with the secondary antibodies Rhodamine Red-X-conjugated AffiniPure Donkey Anti-Rat IgG  and Alexa Fluor 488 donkey anti-mouse . Sections were finally rinsed in TBS , mounted onto gelatine-coated slides, air-dried and coverslipped with Aqua-Poly/Mount .For double immunofluorescence staining the brain sections were pretreated in the same manner as described for immunoperoxidase staining omitting the HPeroxidase staining was performed on every sixth section (in order to avoid counting of neurons twice) and the total number of BrdU-positive cells was counted in the complete dentate gyrus with an Axioplan 2 microscope . The number of BrdU-positive cells was multiplied by 6 to calculate the total number of BrdU-positive cells in the whole dentate gyrus as it was reported in previous studies t-tests were used to describe differences between groups at different time points whenever ANOVA revealed a significant GROUP × TIME interaction. Statistical analysis of cell counts in the DG in the different experimental groups was performed using a one-way analysis of variance (ANOVA) followed by post-hoc t-tests (Tamhane-T2). The IC-wt-group was excluded from the one-way ANOVA due its small n-number but compared to the IC group using a t-test for unpaired samples. Furthermore, we performed some correlation analyses between the number of BrdU-positive cells and the parameters swelling, mechanical hyperalgesia at the inflamed knee joint and asymmetry in weight bearing using Pearson correlation analysis. P-values <0.05 were considered significant.SPSS 19.0.0.1 software  was used for statistical analysis. Data were first tested for normal distribution using Kolmogorow-Smirnow tests. For comparison of the behavioral data between groups, repeated-measures ANOVA was applied with the between-subjects factor GROUP  and the within-subjects factor TIME . Post hoc The injection of mBSA into the knee joint of immunized rats induced a pronounced unilateral inflammation (AIA), similar as in previous experiments Application of pressure onto the knee evoked a withdrawal response. In immunized rats the withdrawal thresholds remained almost stable. But compared to IC rats the AIA rats showed a significant lowering of withdrawal thresholds , with a Using videoradiography, we documented the locomotor behavior  = 19.858; p<0.001). The control group without any treatment (NC) exhibited 1990±283 cells (n = 7 rats). Seven rats which just underwent irradiation with X-rays (CXray) exhibited on average 2609±151 BrdU-positive cells which is not different from the number of proliferated cells in NC rats (p = 0.414). Rats which were only immunized  showed 3957±233 cells, and this number is significantly higher than BrdU-positive cells in either the NC or the CXray group (p<0.01). AIA rats  exhibited 4155±218 BrdU-positive cells which is not different from the number in immunized rats. Thus the main increase of BrdU-positive cells was observed after immunization, and no further increase was found during arthritis.After the in-life phase of 42 days, BrdU-positive cells in the DG were quantified in the 5 experimental groups. In addition, 3 rats (IC-wt) were immunized but they were not tested for changes in pain- and locomotor behavior. Thus they can be directly compared to the IC group to assess the influence of behavioral testing alone on the total number of BrdU-positive cells. The column on the right in  = 8.339; p<0.001). Almost all BrdU-positive cells in AIA and IC rats were also NeuN-positive (97% in AIA rats and 95% in IC rats), in the NC- and CXray group 91% of BrdU-positive cells were NeuN-positive which is slightly but still significantly less than in the AIA rats. Essentially most BrdU-positive cells were neurons.To identify BrdU-positive cells 42 days after the first BrdU-injection, 30 BrdU-positive cells of the DG were analyzed in each animal for coexpression of the neuronal nuclei marker NeuN. A specimen of immunofluorescent double-staining of cells in the DG is shown in The present study addressed neurogenesis in the dentate gyrus during antigen-induced arthritis in the adult rat. Specifically we investigated whether changes of neurogenesis can be attributed to inflammation and/or to the arthritis-induced neurological symptoms pain and altered locomotion. We found significantly enhanced neurogenesis in rats with unilateral AIA which exhibited significant mechanical hyperalgesia at the inflamed knee joint and pronounced alteration in their locomotor behavior. However, neurogenesis was similarly enhanced in rats which were only immunized but in which no AIA was induced and which showed normal locomotor behavior and no pain. Thus, in the present study enhanced neurogenesis was significantly associated with the immunization process and neither with manifest additional local inflammation alone nor with significant alteration of locomotor behavior and hyperalgesia at the inflamed knee.A possible confounding factor of the study was the use of X-rays because X-irradiation may reduce the proliferation of neuronal precursor cells from the subgranular zone in the DG Compared to non-immunized controls (NC and CXray) the total number of BrdU-positive cells was more than doubled in both the immunized and the AIA rats, and the percentage of BrdU/NeuN-double-positive cells was also elevated in both groups. Immunized rats and AIA rats both received BrdU injections three weeks after the first immunization, at the time point at which the antigen was injected into the knee in the AIA group. Because AIA develops rapidly within several hours and shows a peak of swelling, hyperalgesia and asymmetry of weight bearing within the first three days of AIA, an effect of local AIA on neurogenesis should be detected by this protocol of BrdU application. However, immunized rats and AIA rats showed similar numbers of BrdU-positive cells. The important conclusion is, therefore, that the development of manifest inflammation did not further stimulate neurogenesis than the previous immunization. The lack of correlation between neurogenesis and swelling (and between neurogenesis and pain-related behavior) supports this conclusion because the numbers of BrdU-positive cells showed a similar distribution in both experimental groups.A previous study investigated neurogenesis in antigen-induced arthritis in mice Interestingly, systemic or intrathecal administration of lipopolysaccharide (LPS) and the ensuing systemic inflammation were shown to be rather detrimental for hippocampal neurogenesis per se. The same applies to the hyperalgesia at the knee joint. The present study does not support a causal relationship between hyperalgesia and enhanced neurogenesis. One could speculate, however, that the immunization-evoked neurogenesis may have facilitated the rapid adaptation of the walking cycle during inflammation, and in this adaptation pain may also be involved.As a consequence of AIA in the knee, rats exhibited pronounced changes both pain-related and locomotor behavior for more than one week. Derived from previous data that exercise and learning increase the proliferation of neural progenitor cells in the dentate gyrus per se does not lead to local inflammation and hyperalgesia. These data suggest, therefore, that immunization and overt pain/stress are producing opposite effects on neurogenesis. The lack of a further increase of neurogenesis after elicitation of AIA in the knee and the concomitant appearance of pain is in line with such a concept.A previous study reported that repeated injection of complete Freund’s adjuvant (CFA) into the paw induced a reduction of neurogenesis similar as chronic stress evoked by repeated immobilization of animals, and it was concluded that persistent pain produces stress-like alterations (inhibition) in hippocampal neurogenesis The factor stress has to be taken into account. Rats were repeatedly anesthetized when they received injections for immunization, AIA induction and BrdU application. Furthermore, rats were transported in boxes to the X-ray system, and they were tested for pain-related behavior and the locomotor pattern. However, the CXray group which was transported to the X-ray system was not significantly different from the NC group which was just kept in the cages. Because stress rather decreases neurogenesis (see above), the increase of neurogenesis during immunization is unlikely to be caused by stress.in vitro administration of IFN-γ to NSCs or neuronal cell lines enhanced neuronal differentiation More likely is a role of certain cytokines which may inhibit or stimulate neurogenesis. Inhibition of neurogenesis was observed for IL-1β, IL-6 or TNF-α all of which are upregulated by LPS In summary, this study shows significant stimulation of neurogenesis in the DG associated with AIA. However, the exploration of the factors which are responsible for the increase of neurogenesis provided the unexpected and surprising result that neither manifest inflammation in the joint nor pain and altered locomotion are responsible for the increase of neurogenesis. Only the process of immunization could be causally linked to enhanced neurogenesis. Thus, this study indicates that an immune process may stimulate neurogenesis even if there are no obvious symptoms of a disease. It seems noteworthy to further explore this novel finding in different aspects. The functional consequences of enhanced neurogenesis upon immunization should be explored. It may be speculated that enhanced neurogenesis contributes to the generation of disease defense mechanism. On the other hand, as shown for recovery following stroke, enhanced neurogenesis can also contain elements of maladaptation. Furthermore it seems attractive to elucidate which mechanisms are at work in the interaction of adaptive immune responses and neurogenesis.


Kinases are often aberrantly activated in various tumor types by overexpression, point mutations, chromosomal translocations and other mechanisms. Therefore, small molecule inhibitors that block the enzymatic activity of kinases have been developed for different targets and are successfully used for the treatment of cancer patients. A major drawback of these molecularly targeted therapies is the frequent development of therapy resistance, often caused by point mutations in the target protein . This cThe hallmark of chronic myeloid leukemia (CML) is the expression of the oncogenic kinase Bcr-Abl, which arises from the Philadelphia chromosome translocation . Bcr-AblRecently, the myristate binding pocket in the C-terminal lobe of the Abl kinase domain was identified as a site for allosteric regulation of Bcr-Abl activity . A smallIn addition, Bcr-Abl as well as its proto-oncogenic constituent c-Abl (ABL1) have a Src homology 2 (SH2) domain N-terminal to their tyrosine kinase domains - a conserved feature among the class of cytoplasmic tyrosine kinases. The analysis of active Abl conformations lead to the finding that SH2 domains are positive allosteric effectors in cytoplasmic tyrosine kinases via the formation of an intramolecular interface of the SH2 domain with the kinase domain . The SH2The exquisite affinity and specificity of monobodies exemplifies its general utility as target validation tools for preclinical studies. On the other hand, the necessity for intracellular delivery that may only be achieved using lentiviral gene transfer or fusion of the monobody to membrane-permeable peptides will likely limit or preclude the use of monobodies as drug-like molecules in clinical applications.In contrast, small-molecule protein-protein interaction inhibitors have been developed for a number of protein targets, breaking with the dogma that protein-protein interfaces are ‘undruggable’. However, interfering with intramolecular domain interactions (like the SH2-kinase interface in Bcr-Abl) might require very high local concentrations of inhibitor that may be difficult to achieve using intracellular delivery of proteins or peptides. Therefore, we believe that the development of a small-molecule inhibitor of the SH2-kinase domain interface that may be used in combination with approved Bcr-Abl tyrosine kinase inhibitors could be feasible.


Lycium barbarum polysaccharides (LBP), extracts from the wolfberries, are good for “eye health” according to Chinese medicine. The aim of our present study is to explore the use of LBP in retinal I/R injury. Retinal I/R injury was induced by surgical occlusion of the internal carotid artery. Prior to induction of ischemia, mice were treated orally with either vehicle (PBS) or LBP (1 mg/kg) once a day for 1 week. Paraffin-embedded retinal sections were prepared. Viable cells were counted; apoptosis was assessed using TUNEL assay. Expression levels of glial fibrillary acidic protein (GFAP), aquaporin-4 (AQP4), poly(ADP-ribose) (PAR) and nitrotyrosine (NT) were investigated by immunohistochemistry. The integrity of blood-retinal barrier (BRB) was examined by IgG extravasations. Apoptosis and decreased viable cell count were found in the ganglion cell layer (GCL) and the inner nuclear layer (INL) of the vehicle-treated I/R retina. Additionally, increased retinal thickness, GFAP activation, AQP4 up-regulation, IgG extravasations and PAR expression levels were observed in the vehicle-treated I/R retina. Many of these changes were diminished or abolished in the LBP-treated I/R retina. Pre-treatment with LBP for 1 week effectively protected the retina from neuronal death, apoptosis, glial cell activation, aquaporin water channel up-regulation, disruption of BRB and oxidative stress. The present study suggests that LBP may have a neuroprotective role to play in ocular diseases for which I/R is a feature.Neuronal cell death, glial cell activation, retinal swelling and oxidative injury are complications in retinal ischemia/reperfusion (I/R) injuries.  Many ocular diseases such as acute glaucoma and amaurosis fugax may be associated with ischemia/reperfusion (I/R). During I/R, there are breakdown of blood-retinal barrier (BRB), accumulation of fluid within retina, oxidative stress and neuronal death Lycium barbarum , a traditional Chinese medicine, has been used for centuries in the East to maintain “eye health and nourish the liver and kidney”, and to balance “Yin” and Yang” in the body Lycium barbarum has a high content of polysaccharides which is approximately 40% by dry mass; therefore studies have been focused to the liquid fraction of the berries, the Lycium barbarum polysaccharides (LBP) The use of animals in this study was conducted according to the requirements of the Cap. 340 Animals (Control of Experiments) Ordinance and Regulations, and all relevant legislation and Codes of Practice in Hong Kong. All the experimental and animal handling procedures were approved by the Faculty Committee on the Use of Live Animals in Teaching and Research in The University of Hong Kong (CULATR #1870-09).C57BL/6N male mice (10–12 weeks old) were used in the present study. The animals were kept in a temperature-controlled room with 12-hr light/12-hr dark cycle in the Laboratory Animal Unit of The University of Hong Kong.LBP extracts were prepared as described previously 2O/30% O2 for induction and 1% halothane in 70% N2O/30% O2 for maintenance) and the rectal temperature were kept at 37±0.5°C. The internal carotid artery (ICA) is one of the bifurcations of the common carotid artery and provides blood supply to the cerebral regions. It also provides blood supply to the eye as the ophthalmic artery is a branch of the pterygopalatine artery (PPA) which originates from the ICA Animals were anesthetized .2. Viable cells in ganglion cell layer (GCL) of the central and peripheral retina were counted. Cells with pyknotic nuclei were regarded as dead cells and were not counted in the counting. Retinal swelling in our I/R model has been shown to be due to the increase in inner retinal thickness Procedures described in our recently published study were adopted 2. TUNEL-positive nuclei in both GCL and inner nuclear layer (INL) were counted.Apoptosis was examined by TUNEL assay  as previously described Antigen retrieval was performed by incubation with proteinase K following deparaffinization of sections. Non-specific binding sites were blocked with normal goat serum for 1 hr at room temperature and then incubated with primary antibodies listed in Sections were blocked with Mouse on Mouse (MOM) blocking solution  and incubated with mouse biotinylated anti-mouse IgG secondary antibody (MOM kit) as previous described The right eye of each animal was subjected to I/R, and the left eye was used as a non-ischemic control. The retinae of right eyes from animals pre-treated with vehicle were compared with those from the non-ischemic control left eyes of both groups. The retinae of right eyes from animals pre-treated with LBP were compared with those from animals pre-treated with vehicle. A double-blinded approach was used in all the experimental procedures, including cell counting and data analyses. Data were presented as mean ± SEM. One-way Analysis-of-variance (ANOVA) followed by the Bonferroni multiple comparison tests  was used in cell counting and IgG extravasations quantification. Kruskal-Wallis Test followed by the Dunn's multiple comparison tests (Prism v4.0) was performed in the semi-quantitative analyses of IHC scores. Statistically significant difference was set at p<0.05.2-hr ischemia followed by 22-hr reperfusion caused severe tissue damage in the vehicle-treated retina. Many cells in the GCL and INL were pyknotic in the central and peripheral retina . The numThere was an increase in inner retinal thickness of the central retina in the vehicle-treated mice as compared with that in the non-ischemic control mice  . On the Ischemia leads to apoptosis in the retina. More TUNEL-positive cells were observed in the GCL of the vehicle-treated I/R retina compared with that of the non-ischemic control retina   and simiPKC-α is a marker for rod bipolar cells. Normally, the cell bodies of PKC-α-positive bipolar cells are located in the outer border of INL and their axons terminate at the inner border of IPL In non-ischemic control retina, calretinin-positive amacrine cells were present in GCL and the innermost layer of INL. The synaptic terminals of amacrine cells formed three distinct strata in IPL . In the In the normal retina, a small population of amacrine cells expresses nNOS In non-ischemic control retina, the expression of GFAP was confined to astrocytes in GCL . In the In the non-ischemic control retina, there was a weak immunoreactivity of AQP4 expressed in the astrocytic end-feet at the ILM and in the perivascular regions at the outer border of INL . The AQPBlood vessel leakage was assessed by IgG extravasations. In the vehicle-treated I/R retina, when compared with the non-ischemic control, there were greater number of blood vessels with immuno-positive IgG signals located outside the endothelial vessel lining. These vessels were situated at the GCL  and OPL Lipid peroxidation was evaluated using PAR immunohistochemistry. In non-ischemic control retina, PAR expression was limited inside the cytoplasm . A profoNT is a marker for nitrosative stress. Minimal NT immunoreactivity was noted in non-ischemic control retina . An incrBRB disruption, glial cell activation, oxidative stress and neuronal death are major concerns in retinal I/R injury. Neuroprotective agents that can retard or prevent these damages are beneficial in treating many ocular diseases in which retinal I/R is a complication. In our present study, we showed that pre-treating mice with LBP for 1 week could protect the animals from I/R injury by reducing neuronal cell death, retinal swelling, glial activation, BRB disruption and oxidative stress.In the present study, unilateral retinal ischemia was induced by occluding the ICA which supplies blood to the ophthalmic artery Lycium barbarum is a dried fruit that is used as a food or a medicine according to Chinese tradition Retinal I/R induces neuronal death in the inner retina, especially RGC. An extensive loss of cells in the GCL has been seen after retinal I/R injury BRB is a protective barrier which consists of the outer and inner BRB. The role of BRB is to maintain the homeostatic condition of retinal microenvironment and exclude harmful substance getting into the retina + and the movement of water inside cells Disruption of BRB leads to swelling of astrocytes and Muller cells processes associated with the activation of GFAP and AQP4 under ischemic conditions Lycium barbarum, increases glutathione peroxidase activity and glutathione levels, and decreases cystine concentrations in retina of rd1/rd1 mice, indicating a decreased level of oxidative stress in retinal generation Oxidative stress plays a role in retinal I/R injury due to the high content of polyunsaturated fatty acid in retina. Reactive oxygen species (ROS) and free radical formation during I/R facilitates lipid peroxidation of membrane, denature of protein and DNA damage Increasing lines of evidence have demonstrated that modulation of immune responses can affect the degenerative processes of neurons in the CNS Our data indicated that pre-treatment with LBP for 1 week could effectively protect the mouse from retinal I/R injury. The present study hence suggests that LBP may be used as a preventive TCM for diseases associated with I/R such as amaurosis fugax and acute glaucoma. Further studies are warranted to evaluate the therapeutic effects of LBP and other potential neuroprotective mechanisms.


Most of our daily actions are selected and executed involuntarily under familiar situations by the guidance of internal drives, such as motivation. The behavioral tendency or biasing towards one over others reflects the action-selection process in advance of action execution . Facing unexpected situations, however, pre-action bias should be withdrawn and replaced by an alternative that is suitable for the situation . To understand the neural mechanism for the counteracting process, we studied the neural activity of the thalamic centromedian (CM) nucleus in monkeys performing GO-NOGO task with asymmetrical or symmetrical reward conditions. The monkeys reacted to GO signal faster in large-reward condition, indicating behavioral bias toward large reward. In contrast, they responded slowly in small-reward condition, suggesting a conflict between internal drive and external demand. We found that neurons in the CM nucleus exhibited phasic burst discharges after GO and NOGO instructions especially when they were associated with small reward. The small-reward preference was positively correlated with the strength of behavioral bias toward large reward. The small-reward preference disappeared when only NOGO action was requested. The timing of activation predicted the timing of action opposed to bias. These results suggest that CM signals the discrepancy between internal pre-action bias and external demand, and mediates the counteracting process—resetting behavioral bias and leading to execution of opposing action. In our daily life, most actions are selected and executed involuntarily, but they are appropriately incited by motivational, habitual or innate drive. For example, when actions are followed by different values of rewards, the highest one among the alternatives tends to be chosen frequently : monkey SJ (5.8–7.5 kg) and monkey MA (6.7–8.0 kg). All surgical and experimental procedures were approved by the Animal Care and Use Committee of Kyoto Prefectural University of Medicine and were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The monkeys had limited access to water for 4–5 days per week, but they received food and water ad libitum on weekends.We used two male Japanese monkeys  and sodium pentobarbital , and supplemental Nembutal  was given as needed. Four head-restraining bolts and two recording chambers were implanted under stereotaxic guidance on the skulls of each monkey. The chamber for recording neuronal activity in the thalamus was positioned vertically over the thalamus. The center of the chamber was positioned midline and adjusted according to Horsley–Clark stereotaxic coordinates (anterior 12–13 mm). The other chamber was not used in this study.We recorded the activity from single neurons that were located primarily in the CM nucleus as well as surrounding thalamic nuclei, such as parafascicular nucleus (PF) and dorsolateral PF (PFdl). Action potentials from single neurons were recorded using tungsten microelectrodes  that were inserted through the implanted recording chamber and advanced by means of an oil-drive micromanipulator . The action potentials were amplified, filtered (50 Hz to 3 kHz) and isolated by spike sorter with a template-matching algorithm . Onset times of the action potentials were recorded on a laboratory computer  together with the onset and offset times of stimuli and the behavioral events such as pressing and releasing the button. In this study, we selectively studied the activity of long-latency-facilitation (LLF) type of neurons, which show burst discharges after unexpectedly presented auditory and/or visual stimuli of long latency , such as knocks on the laboratory door. We also recorded licking movement by means of a strain gauge  fixed to the waterspout.Analysis of behavioral and spike data and statistical test were performed using a Visual Basic  and R statistical computing environment .t-test. Reaction times  and movement times  in GO trials were computed and compared between bias conditions by two-sample t-test.For behavioral data analysis, we excluded the data of the initial eight correct trials during the transitional phase between blocks of trials with different action-reward associations. Error rates for each trial type were calculated in each block, and were averaged across blocks in each bias condition. The average error rate for each trial type was compared between bias conditions by two-sample Background: the 250-ms period (500–750 ms) before pressing the hold button; and (2) Post-instruction: the 250-ms period (250–500 ms) after instruction onset. The statistical significance of changes in the discharge rate of the post-instruction activity for each of four trial types was evaluated by two-sample Wilcoxon test (p < 0.05) compared to the background activity. To quantify the preference of neural response, we performed receiver operation characteristic (ROC) analysis. For this analysis, we counted the number of spikes in the post-instruction period for each trial and constructed the distribution of spike numbers for each of GO(+R), GO(−R), NOGO(+R), and NOGO(−R) activity. Then we calculated the area under the curve of the receiver operating characteristic  using a distribution set . The ROC value gives us the general measure of selectivity; 0.5 indicates no preference while 0 and 1 indicate large- and small-reward preference, respectively. We examined the relationship between the latency of peak activation after GO instruction and RT in the same trial for each LLF neuron. First, we determined the peak activation after GO, although it was not detected in the remaining trials mostly because of the absence of spikes. To examine the relationship between neuronal activation and RT, we performed linear regression analysis on a trial-by-trial basis. For each trial, we determined the peak of activity  smoothed with a Gaussian kernel (SD = 20 ms) during the period from the onset of GO instruction and 100 ms after GO reaction. Latency and magnitude of peak activity were used as regressors for multiple linear regression analysis of the GO RT.For spike data analysis, we excluded the data of error trials and retrials after error trials, as well as eight successful trials after the block transition. Based on the previous study, we examined the discharge rates of each recorded neuron during two task epochs: (1) At the end of all recording experiments, small electrolytic lesions were made at 8 and 16 locations along selected four and eight electrode tracks in monkeys SJ and MA, respectively. Direct anodal current (20 μA) was passed for 30 s through tungsten microelectrodes. After all studies were completed, the monkeys were deeply anesthetized with an overdose of sodium pentobarbital , and perfused with 4% paraformaldehyde. Half of the coronal 50-μm-thick sections were stained with cresyl violet (Nissl). For monkey SJ, the other half of the sections were stained by thiocholine method to demonstrate acetylcholinesterase (AChE) activity. The anatomical boarders of thalamic nuclei were assessed on histological sections by referencing the histological criteria of the monkey thalamus in conjunction with the assessment of their AChE activity. Histological reconstruction of the microelectrode tracks in relation to the electrolytic lesion marks allowed us to verify the location of the neuronal recordings.p < 0.001, t-test, Figure p < 0.001, t-test). The monkeys made an error  more frequently in small-reward trials than in large-reward trials . These results suggest that, while large-reward action is facilitated by internal motivational drive, slowing of small-reward action is due to the conflict between internal bias and the external demand to overcome to it.Two macaque monkeys performed in biased blocks of GO-NOGO task. Both average RTs and average MTs were significantly shorter in GO(+R) trials than in GO(−R) trials in both monkeys  while the monkeys performed in a biased block of GO-NOGO task. LLF neurons were identified as showing burst discharges after unexpected auditory and visual stimuli with long latencies . Collectively, these results indicate that LLF neurons preferentially respond to instruction for an action associated with a smaller reward.Figure r = −0.59, p < 0.001; monkey MA, r = −0.40, p < 0.001; Figure In GO-NOGO task, the action-reward association was stable within a block of trials (GO(+R)/NOGO(−R) or NOGO(+R)/GO(−R); 40–120 trials), inducing a behavioral bias as shown above. However, even under the same action-reward association, the degree of behavioral bias varied block-by-block. For example, the median RT of GO(+R), an index of behavioral bias of a block, ranged from 307 to 430 ms, and from 204 to 297 ms, in monkeys SJ and MA, respectively. This gave us the opportunity to test whether the LLF preference of small reward is modulated by the strength of behavioral bias; assuming that recorded LLF neurons were sampled from a homogeneous population, the small-reward preference would be stronger when the neuron was recorded under stronger behavioral bias. To test this, we examined a block-by-block relationship between NOGO(−R) preference of the LLF response and the median RT of GO(+R) action. As shown in Figure r = −0.54, p < 0.0001), but not in monkey MA .We also examined the GO(−R)-NOGO(+R) block, where RT in GO(−R) trials was affected by the balance between pre-action bias and its counteracting. In this case, we could not find a consistent relationship; a significant negative correlation between GO(−R) preference and median RT was observed in monkey SJ . We detected a peak response in average 68% of GO(−R) trials, and defined the magnitude and latency of the peak activity  between peak latency of GO(−R) activity and RT  indicated that the peak of LLF activity was constantly preceded to the following action  between peak magnitude and RT . We detected peak activity in relatively fewer trials (average 39%). We found significant correlation (p < 0.01) between peak latency of GO(+R) activity and RT less frequently . In even-reward block, 9/16 neurons showed significant GO response. Peak latency was detected in an average 56% of trials. Significant correlation was found in 7 of 9 neurons. Together, the timing of LLF discharges can predict the timing of the following action, and especially action that has not been biased.We performed the same analysis on 35 LLF neurons that showed significant GO(+R) response in GO-bias block , median peak latency was significantly longer than the median RT of GO(+R) trials . These results suggest that CM makes little or no contribution to the suppression of biased action.Similarly, we examined the timing of LLF activity after NOGO(−R) instruction in GO-bias block. In Figure In the present study, to investigate the neural mechanisms for counteracting pre-action bias, we tested monkeys performing GO-NOGO task, in which either GO or NOGO action was associated with large reward. The monkeys responded to instruction for large-reward action quickly and correctly, but reacted slowly to instruction for small-reward action. This suggests that, while large-reward action is facilitated by virtue of internal motivational drive , slower small-reward action is due to a conflict between internal drive and external demand to overcome it . LLF neurons, a subpopulation of neurons located mainly in the CM nucleus, exhibited phasic burst discharges after GO and NOGO instructions especially when associated with small reward. We found that the small-reward preference of the LLF response was positively correlated with the strength of behavioral bias toward large reward. A similar preference-bias relation was found in the block where both GO and NOGO actions were rewarded equally. When only one action type  was requested with either large- or small-reward outcome, the small-reward preference disappeared. Furthermore, there was a positive temporal relation between LLF activation to GO(−R) instructions and the following GO(−R) actions on a trial-by-trial basis. LLF activations to NOGO(−R) instructions did not precede GO(+R) actions in the same block. Taken together, the results provide a better understanding of the role of CM in counteracting pre-action bias; CM neurons detect and signal external demand to overcome preset bias according to the degree of the bias.As shown in a previous study  and GO(SLOW) in Figure in vitro slice study  responses were not always ahead of time for initiation of GO(+R) action . Neurons in the caudate nucleus exhibit pre-movement activity that would create a motivational bias toward the contralateral space  in Parkinson’s disease (PD). Specific and remarkable (30–40%) neuronal loss in the CM-PF complex was demonstrated by postmortem brain studies in PD patients (Henderson et al., In summary, the present data demonstrated that neurons in the thalamic CM nucleus respond to external demand of action opposed to behavioral bias and signal the discrepancy between external demand and pre-action bias, the occurrence of which is followed by opposing action. The CM discrepancy signal may be used in its main target structure, the posterior putamen, to overcome its activity for the preset bias. This counteracting process seems to enable one to execute the opposite action, which is demanded externally but is not yet internally motivated or prepared. Interrelations between the basal ganglia and the thalamic CM-PF complex thus may allow us to switch our behavior properly and flexibly.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Molecular imaging of breast cancer has undoubtedly permitted a substantial development of the overall diagnostic accuracy of this malignancy in the last years. Accurate tumour staging, design of individually suited therapies, response evaluation, early detection of recurrence and distant lesions have also evolved in parallel with the development of novel molecular imaging approaches. In this context, positron emission tomography (PET) can be probably seen as the most interesting molecular imaging technology with straightforward clinical application for such purposes. Dozens of radiotracers for PET imaging of breast cancer have been tested in laboratory animals. However, in this review we shall focus mainly in the smaller group of PET radiopharmaceuticals that have lead through into the clinical setting. PET imaging can be used to target general metabolic phenomena related to tumoural transformation, including glucose metabolism and cell proliferation, but can also be directed to specific hormone receptors that are characteristic of the breast cancer cell. Many other receptors and transport molecules present in the tumour cells could also be of interest for imaging. Furthermore, molecules related with the tumour microenvironment, tumour induced angiogenesis or even hypoxia could also be used as molecular biomarkers for breast cancer imaging. PET is a molecular imaging modality in which compounds labelled with positron emitting radioisotopes are used to measure biological processes with only trace amounts of the radiolabelled probes. Molecules labelled with positron-emitting radionuclides are retained in tissues as a result of binding to a receptor, or cell entrapment owing to enzyme-catalyzed conversion after uptake by a cell membrane transporter. Tomographic images of the distribution of the radioactivity within the body can be generated and quantitatively evaluated by coincidence detection of the gamma rays resulting from the mutual annihilation of a positron emitted by the radionuclide and an electron of a nearby atom.PET radiotracers are molecular probes that can be designed and synthesized to target very many different molecular and cellular events. In the specific case of breast cancer, there is a wide panoply of specific and general targets one could think about. However, being there a great number of molecules labelled with different positron emitting radionuclides see  that havin vivo largely exceeds 300, only a fraction has been used in humans. In addition, probably more than 90% of the clinical PET studies are performed with the analogue of glucose 2-[18F]-fluoro-2-deoxy-D-glucose (FDG). Uptake and metabolism of FDG within tumour cells is higher than in normal tissue because of increased glycolysis. The incorporation of FDG to the neoplastic cells is favoured by the increase of glucose membrane transporters (GLUTs) which are overexpressed in breast cancer due to the activation of their genes. FDG is phosphorylated to FDG-6-phosphate by hexokinase-1, which also has increased activity in tumour cells due both to genetic and allosteric modifications. Therefore, the detected concentration of labelled FDG is proportional to the uptake and metabolism of glucose.Although the number of PET tracers synthesised and tested In patients with breast cancer, FDG PET/CT results in improved sensitivity for the detection of both lymph node infiltration and distant metastases compared to conventional imaging techniques . TherefoTumour uptake of FDG is variable and depends not only on tumour size, but also on the histological type and histological grade. The hormone receptor status and other immunohistochemical factors with prognostic value are also relevant, such as p53 and Ki-67 expression. FDG uptake is higher in ductal carcinoma than in lobular carcinoma and in patients with poor prognostic features as high grade and hormone receptor negativity fluorothymidine (18FLT) as a biomarker for in vivo imaging of cell proliferation, demonstrating that it was resistant to degradation in vivo, retained in proliferating tissues by the action of thymidine kinase 1 and produced high-contrast images of normal marrow and tumours.Back to 1998, Shields et al. fluoromisonidazole (18FMISO) methyl-choline (11C-choline) were established, Contractor et al. -fluoro-17β-estradiol (18F-FES) in a set of 13 patients with primary breast tumours. They found an excellent correlation between uptake of 18F-FES and estrogen-receptor concentration measured in vitro after excision of the lesions.Already back to 1988, Mintun et al.  pioneere18F-FES can be seen as a unique method to noninvasively obtain molecular information of ER expression. No other procedure can provide information on a whole body basis of the ER status in metastasic breast cancer. It is well known that ER expression at metastasic sites might not be the same as the ER expression at the primary disease due to relatively common phenotypic changes. In any case, loss of ER expression is more common than gain of ER expression.Nowadays, whole-body PET with 18F-FES PET can reliably detect ER-positive tumour lesions and that 18F-FES uptake correlates with ER expression as measured by immunohistochemical methods. Furthermore, low 18F-FES uptake has been reported to be a strong predictor for failure of antihormonal therapy. For a comprehensive review of 18F-FES PET, see FPPRGD2 have also been recently tested in humans [αvβ3 integrin have been developed in the last years and tested in vivo in small animals, including some very promising gallium-68 labelled RGD peptides [Other RGD-derived radioligands based on the dimeric RGD moiety such as [n humans . In addipeptides .in vivo behaviour and efficacy of monoclonal antibodies (mAbs) in individual patients. Very many papers have been written to describe the benefits of these visible magic-bullet approach—see [Immuno-PET has largely been seen as an exciting option for better understanding the oach—see , 46 and For labelling intact antibodies with a low-clearance kinetics, longer-lived radioisotopes such as zirconium-89 or iodine-124 are the election of choice, while for radiolabelling mAbs fragments or constructs  which are more rapidly cleared from the body, shorter-lived positron emitters such as gallium-68 or copper-64 might be ideally suited.89Zr-immuno-PET in clinical mAb development and applications. The aforementioned advances are related with the production and commercial availability of 89Zr for clinical use and the development of chelates for facile and stable coupling of 89Zr to mAbs.Currently, 12 mAbs have been approved by the FDA for the treatment of cancer, all being intact mAbs . Seven oin vivo. A 68Ga-labeled HER2 derivative affibody has been used to monitor HER2/neu expression in breast cancer xenografts [in vivo. The mAb trastuzumab targets the human epidermal growth factor receptor kinase (ERBB or HER) signaling network and has a history as a therapeutic for metastatic or adjuvant treatment of oncological disease. Upregulation of the ERB2 (HER2/neu) receptor has been associated with metastasis and poor prognosis in many cancers, including breast cancer. Specifically, HER receptors stimulate growth and regulate survival and differentiation. Because of variable HER2/neu receptor expression over time, noninvasive and dynamic measurement methods would be ideal for monitoring potential treatment and disease prognosis.Overexpression of HER2/neu in breast cancer is correlated with a poor prognosis. It may vary between primary tumours and metastatic lesions and change during the treatment. Therefore, there is a need for a new means to assess HER2/neu expression nografts  and prel89Zr-trastuzumab allows visualization and quantification of uptake in HER2-positive lesions in patients with metastatic breast cancer. The PET images obtained with 89Zr-trastuzumab revealed high spatial resolution and a good signal-to-noise ratio. Excellent tumour uptake and visualization of HER2-positive metastatic liver, lung, bone, and brain tumour lesions were obtained. 89Zr-trastuzumab PET visualized bony metastatic disease. These early studies show great promise for the potential of 89Zr-trastuzumab in immuno-PET.However, there is only one paper published so far describing the use of immuno-PET in humans and just in fourteen patients  and an aA number of different tracers targeting diverse cellular biochemical mechanisms involved in breast cancer have been developed, synthesised, and tested in breast cancer xenograft models. Although an exhaustive list is well beyond the scope of this paper, it is worth citing a couple of very recent articles describing the use of sugar-like derivatives as alternatives to FDG for tumour imaging: a fluorine-18-labelled inositol derivative  and a fl