GST and NQO1, both of which may infer risk of benzene-induced MDS. Polymorphisms in the tumor suppressor gene, TP53, and the inflammatory cytokine gene, TNF-α, were also noted, albeit inferring opposing outcomes. The alleles identified in the DNA repair gene RAD51 indicated an increased risk for MDS in MDS patients and low blood cell counts in benzene-exposed workers. We propose the weight-of-evidence approach as a tool to assist in organizing the sea of emerging molecular data in exposure-disease paradigms.Information on polymorphisms, mutations, and epigenetic events has become increasingly important in our understanding of molecular mechanisms associated with exposures-disease outcomes. Molecular landscapes can be developed to illustrate the molecular characteristics for environmental carcinogens as well as associated disease outcomes, although comparison of these molecular landscapes can often be difficult to navigate. We developed a method to organize these molecular data that uses a weight-of-evidence approach to rank overlapping molecular events by relative importance for susceptibility to an exposure-disease paradigm. To illustrate the usefulness of this approach, we discuss the example of benzene as an environmental carcinogen and myelodysplastic syndrome (MDS) as a causative disease endpoint. Using this weight-of-evidence method, we found overlapping polymorphisms in the genes for the metabolic enzymes  In an idealistic view of carcinogenesis, the molecular initiation of a disease process can be directly tied to a genetic mutation or chromosome event caused by environmental exposure to a carcinogen. However, a single event is usually insufficient to induce cancer and other secondary events such as additional gene mutations and/or chromosome changes are usually required . These aFurther, in an idealistic view of a cause and effect relationship between an environmental carcinogen and disease endpoints, exposure to the environmental carcinogen would remain evident at the time that the disease manifests. For example, a causal link has been established between environmental exposure to aflatoxin, DNA modification, and the manifestation of liver cancer . UnfortuHerein, we asked the following: if exposure to a particular environmental carcinogen is implicated in the cause of a disease but the exposure is not evident at the time of disease diagnosis, what additional molecular events  can be linked to the exposure-disease paradigm? Further, what additional mutational events can be linked to disease progression, as not every and not all exposures lead to disease outcomes? We propose that a “weight-of-evidence” (WoE) approach can be applied to compile evidence from multiple sources in the published literature to create a molecular landscape for the environmental carcinogen and for the disease endpoint in question. Herein, we describe the proposed WoE approach for navigating the molecular landscapes of the exposure-disease paradigm. We discuss this application in the context of benzene-induced myelodysplastic syndrome (MDS), to demonstrate how this approach can be used to identify and assign relevance to molecular events associated with both exposure and outcome, taking the multistep process of carcinogenesis into consideration. Findings from this approach may be useful in identifying a biomarker for a specific environmental chemical, identifying a molecular endpoint to be used in future molecular epidemiology studies, providing support for traditional epidemiology in establishing causal inference, and/or identifying molecular events that are important for those individuals susceptible to a specific exposure-disease paradigm.sustainable cellular growth independent of a growth signal,abnormality in proteins that regulate the cell cycle,loss of the ability to respond to programmed cell death,cellular immortalization marked by the retention of telomeres,continuous blood supply through sustained angiogenesis,loss of adhesion ability resulting in tissue invasion and metastasis.The interval between the exposure to a carcinogen and the manifestation of disease allows time for these molecular changes to occur. Latency is technically defined as the period of apparent inactivity between the time of first exposure to a causative agent and the time for response, or the first clinical manifestation of the disease [While there are multiple types of mutations identified in cancers, it has been suggested that frank carcinogenesis occurs only when cells acquire defects in the following six key areas of cellular control :sustaina disease , 5. The  disease . Latency disease . Thus, c Monographs on the Evaluation of Carcinogenic Risk to Humans [WoE refers to the interpretive methods commonly applied to bodies of literature when conducting hazard and risk assessments. These types of approaches have been used for decades by academics, practitioners, and regulatory agencies in both formal and informal risk assessment processes to establish understandings of causality, discuss chemical hazards, and set regulatory action levels for exposure or contamination. For example, the International Agency for Research on Cancer (IARC) applies a WoE approach to evaluating carcinogenic risk, which they describe in the preamble to theiro Humans , 8. Simio Humans , 10. in vitro and in vivo toxicology, various epidemiological designs, and molecular studies), considering broader aspects of quantitative study quality [Yet, while WoE is not a new concept, scientists have faced challenges in the fact that the meaning of “weight-of-evidence” in its practical application is not necessarily clear or well defined . Recentl quality . WoE app quality  proposedHerein, we suggest another application of WoE, which combines traditional methods for establishing causal inference with more recent uses in providing direction for molecular research. While no interpretive WoE method can eliminate the need for some level of expert judgment, WoE frameworks such as the one we suggest herein can help reduce subjectivity and increase transparency in the vast body of literature, which can lead to greater understanding of a particular exposure-disease paradigm than might be possible without the aid of such a tool.Benzene is a known environmental carcinogen given adequate dose and duration of exposure . Crude oBenzene is also a known leukemogen but the molecular events required for the development of benzene-induced leukemia occur over an 8- to 15-year latency period , 21. As Recent investigation has revealed that MDS is the most sensitive dose-dependent carcinogenic endpoint following benzene exposure in the occupational setting, with MDS occurring in some petroleum workers at peak exposure levels as low as 3 parts per million (ppm) . MDS rep s-phenylmercapturic acid (sPMA), benzene-induced depression of peripheral blood parameters , and adducts to hemoglobin and albumin [Regarding markers for benzene-induced disease, several biomarkers of exposure to benzene exist, including urinary metabolites of benzene albumin –28. Whil albumin , 24. TheIn an effort to examine molecular events associated with MDS disease progression, large scale genome-wide associated studies have been conducted with hundreds of MDS patients –31. In tIn considering the molecular landscapes, it is important to differentiate a clear definition for each molecular piece of the landscape. Polymorphisms are inherent to the genome and were originally used to describe variations in shape and form that distinguish normal proteins within a species from each other which makes each human genome unique. Because polymorphism can cause extreme variation in protein function, some polymorphisms can infer genetic susceptibility to a certain agent or disease outcomes whereas other polymorphisms can infer resistance. A mutation, conversely, is a permanent change of the DNA sequence following an exposure or event. Mutations result from unrepaired damage to DNA that occurs due to errors in the process of replication or from the insertion or deletion of segments of DNA by exogenous agents. Finally, epigenetic events are those that cause modification of the genome without modification of the DNA itself. Epigenetic events can result in a change in the expression of the underlying genetic trait by altering the timing and quantity of expression at key points in time .We developed a WoE approach as a tool to navigate the molecular landscape of any given exposure-disease paradigm. The approach is demonstrated in The first tier of the WoE approach involves collecting baseline information on the outcome and exposures of interest. The goal of this tier is to determine if enough baseline evidence exists to illustrate that a relationship may exist between an exposure and outcome to warrant more detailed, intensive molecular investigation. Without documented indications that the outcome of interest may be associated with the suspected environmental carcinogen, and that exposure to that agent likely occurred or is occurring in the study population, molecular investigations related to those exposures and outcomes are not a useful tool. The first step within this tier is to perform a literature search to determine whether the disease endpoint in question has been associated with the environmental carcinogen of interest in published, peer-reviewed literature. This can include both epidemiological and toxicological studies, though one should generally be cautious of study quality and consider the overall strength of the body of literature on this association. Simultaneously, one should determine whether the individual or group of individuals in question had a historic exposure or potential/likely exposure to a suspected toxic agent prior to the development of the disease endpoint. At the most simplistic level, this can be achieved through establishing work history summaries or definitions of cohort groups based on job categories . More detailed, specific methods to establish an exposure history should also be employed if data is available, such as exposure monitoring or industrial hygiene records. Additionally, if exposure to the environmental carcinogen was documented at the time of alleged exposure, records indicating biological evidence of exposure  add an additional level of certainty. An understanding of available biomarkers of exposure to the exposure agent and the residence time of those markers following exposure cessation is also useful at this time.Once there is an understanding of a reasonably assumed exposure that occurred in the study population as well as evidence of an association between the exposure agent and the outcome, one can confidently move into the second tier of the WoE approach. This tier involves conducting a literature search to establish the molecular landscape associated with the exposure-outcome paradigm of interest. A comprehensive, systematic literature search should be conducted to identify peer-reviewed published literature related to molecular events  for both the environmental carcinogen and the disease endpoint. Identified information on the molecular event and the function or effect of that event can be organized into lists or tables, such as those shown in Tables The goal of the third tier is to compare the molecular landscapes for both the environmental carcinogen and the disease endpoint identified during the second tier. To do this, the pieces of the molecular landscapes should be compared side by side to identifying overlapping molecular events. These overlaps can occur in a number of ways. For example, is there a single nucleotide polymorphism (SNP) on the exposure side that represents the same change in protein function as the SNP from the outcome side ? Or, on the other hand, are there SNPs that are counterbalanced across the exposure and outcome sides? Direct overlaps of the same molecular events on both the exposure and outcome side represent the highest priority for further investigation in the fourth tier. Counterbalanced or complementary overlaps that result in counterbalancing functions represent a second priority level for the fourth tier. Finally, there may be mutational pieces seen on the exposure side that result in a functional change related to the target organ in which the outcome is seen. Even if a similar or counterbalanced/complementary piece is not seen on the outcome side, these molecular pieces could be of interest and should be carried into the fourth tier of the WoE approach. If comparison of the molecular landscape at the tier 3 level reveals no overlaps, one must consider whether this implies that the molecular landscape has not yet been fully developed, in which case more researches into the molecular changes associated with the agent of exposure and/or the disease endpoint are warranted, or that the exposure-disease association of interest is not supported by the molecular landscapes.Finally, once the molecular landscapes have been narrowed down to only the overlapping pieces, the fourth tier of the WoE approach can be implemented. This tier involves ranking overlapping pieces of the molecular landscape by value of the functional protein affected by that molecular event in the disease process. When investigating exposure and effects, the priority for the functionalities is determined based on the importance of that change to susceptibility to carcinogenesis/disease progresses and the specificity that the change implies for the specific exposure-disease paradigm versus a generic change that is seen in all cancers.activation and deactivation enzymes of environmental carcinogens,proteins that function specifically in the target organ of toxicity,proliferation and inflammatory cytokines,DNA repair enzymes,tumor suppressor proteins,proteins responsible for cell adhesion.Similarly, we suggest an initial priority level epigenetic event as follows (from greatest to least relevant): hypo- or hypermethylation (modify timing of DNA expression into protein),hypo- or hyperacetylation (modify DNA expression into protein),histone modification (open DNA reading frame).Base on our professional judgment, we suggest an initial priority level for polymorphisms and mutations as follows (from greatest to least relevant):The result of the fourth tier, as well as the overall result of the WoE approach, is a list of related and relevant molecular events associated with the exposure and the outcome side of a given exposure-disease paradigm that can be used to indicate a more likely association has occurred between that exposure and the outcome. These molecular events warrant the highest level of further consideration within the sea of information related to the given relationship.We suggest that common polymorphisms, genetic mutations, and epigenetic events be given the highest WoE ranking if the environmental carcinogen in question is directly or indirectly toxic through DNA adduct formation , and then DNA repair mechanisms should be elevated, as genetic changes in DNA repair genes would be expected in the process of carcinogenesis in any tissue . We antiTo illustrate how this WoE approach works, we applied the methods to the exposure-disease paradigm of benzene and MDS. In this specific scenario, we chose to rank the overlapping molecular landscape on the basis of susceptibility to benzene-induced MDS. We started our WoE approach at the second tier, because the first tier step of determining whether evidence exists for an assumed association between benzene and MDS has been done by other researchers, and this was a hypothetical exercise so there was no individual or cohort for which exposure assumptions needed to be established . A literThe known molecular landscapes for MDS and benzene, respectively, as determined by our literature search are displayed in Tables Where possible, we used the published literature to determine if the molecular events identified for benzene and MDS represented the same type of change in protein function . Molecular landscapes  for MDS and benzene ranked by the WoE approach are shown in S-transferase (GST)  and NAD(P)H:quinone oxidoreductase 1 (NQO1). We also found additional polymorphisms in benzene-exposed workers in cytochrome P4502E1 (CYP2E1) and myeloperoxidase (MPO) but not in MDS patients. These metabolic enzymes have been examined in assays for variations in activity associated with benzene toxicity [ para-benzoquinone (pBQ). Detoxification of benzene metabolites in the liver is controlled by GST genotypes whereas detoxification of pBQ in the bone marrow is thought to occur through the NQO1 enzyme [ NQO1 germline polymorphism C609T, which results in a lowering of NQO1 enzyme activity, which in turn may result in an increase in susceptibility to MDS in benzene-exposed individuals. However, a NQO1C609T polymorphism has been shown to have no effect in MDS [ GSTT1 and GSTM1 are genetic polymorphisms of GST in humans, and a homozygous deletion in these enzymes leads to a complete absence of enzyme activity [ GSTM1 and NQO1 polymorphisms rank as R = 1 and R = 2, respectively, on the WoE scale.By overlapping the molecular landscapes for MDS and benzene, we identified common polymorphisms and ranked them by molecular event of interest (MEoI) based on the criteria outlined in Methods. Our finding showed overlap in the genes for the metabolic enzymes glutathione-toxicity . For exa1 enzyme . We fount in MDS . In addiactivity . It was activity . In an eactivity . Taken tactivity . Therefo TP53 have been shown to be frequent mutations in human cancers [ TP53 common genetic polymorphism was ranked as R = 4 on the WoE scale.Mutations in the tumor suppressor gene cancers . Several cancers . This su cancers . Becauseα) protein is a major regulatory cytokine that plays a role in many immune-mediated diseases and hematologic malignancies [ TNF-α genetic polymorphism, which increases the transcription level of this inflammatory cytokine, was associated with MDS patients [α protein may also be responsible for promoting a proinflammatory state in benzene-exposed workers; one study showed that only the −238 TNF-α and not the −308A TNF-α polymorphism was significantly associated with the development of benzene-induced dysplasia and not with an increased risk of MDS [α and −238 TNF-α showed the opposite effect in these studies, TNF-α does play a significant role in the target organ of toxicity [α would also rank as R = 2 on the WoE scale.Tumor Necrosis Factor alpha  counts lower than 4000 μL [ RAD51 polymorphisms have been associated with multiple diseases so this lowers the rank to R = 3.The RAD51 protein plays an important role in DNA repair, meiosis, chromosome segregation, and chromosome stability, and its dysregulation has been associated with multiple diseases . A meta- 4000 μL . Since t RUNX gene, also known as AML1, codes for an important transcription factor, “core binding factor” alpha subunit, which is a transcription factor that regulates commitment to erythroid and granulocytic lineages and initiates the terminal differentiation of the myeloid lineage [ RUNTX1T1 (ETO) in a chromosome translocation t, which is one of the most frequent karyotypes in AML [ RUNX gene mutations have been identified in some cases of MDS and generally infer unfavorable prognosis [The lineage . AML1 iss in AML . RUNX gerognosis , 31. RUNX methylation status was examined in a cell line treated with the benzene metabolite HQ, researchers found that HQ induced hypermethylation in RUNX as well as hypomethylation in RUNTX1T1 [ RUNX1 in some MDS patients and the RUNX1 epigenetic event shown in in vitro cell culture treated with benzene metabolites may be important if the RUNX1 finding can be reproduced in workers exposed to benzene, especially since this mutation occurs in the target organ of toxicity. We propose that the RUNX1 mutation in MDS and epigenetic changes shown in an in vitro cell culture treated with benzene metabolites rank as R = 5 on the WoE scale.Interestingly, whenRUNTX1T1 . Taken t DNMT3A gene expression [ DNMT gene mutation ranks as R = 6 on the WoE scale.DNA methyltransferases (DNMTs) play a key role in establishing and maintaining methylation. DNA methylation is considered to be the initial step in establishing the inactive chromatin state and is critical for maintaining silence  in protooncogenes . Reducedpression . The epiR = 7 and R = 8, respectively.Polymorphisms in the metabolic enzyme myeloperoxidase (MPO) within the bone marrow, specifically, the balance between MPO and NQO1 and the benzene metabolites, have been studied. Because both of these polymorphic enzymes occur in the target organ for benzene toxicity, they may have a significant effect on susceptibility to benzene toxicity . Interle GPA alleles were examined in a small group of benzene-exposed workers and control subjects, it was shown that lifetime cumulative exposure to benzene was associated with the NN variant of GPA but not with the N0 variant. It was suggested that the NN mutation occurred in longer-lived bone marrow cells and that the NN variant resulted from a loss of the GPA M allele, possibly through benzene-induced duplication of the N allele. The N0 variant was presumed to occur through point mutations or deletions [R = 9.The glycophorin A (GPA) locus codes for an erythroid lineage specific protein with two allelic forms (M and N). Wheneletions . AlthougWe developed this WoE process in an effort to understand the commonalities between the molecular landscape of an environmental carcinogen and the molecular landscape of a known or suspected disease outcome. We showed how the WoE approach could be used to identify and assign relevance  to overlapping genetic information associated with susceptibility in an exposure-disease paradigm. We envision that this approach can be modified to identify and rank the most relevant overlapping epigenetic events in an exposure-disease paradigm.On an individual level, the approach might have practical application for purposes of prevention such as identifying worker susceptibility in an occupational setting. For example, an individual with the potential for high occupational exposure to a specific chemical with a known association with a specific disease outcome could undergo molecular testing to determine whether they are (a) susceptible to initial toxicity from exposure to a specific chemical, (b) susceptible to initiation of molecular events associated with disease progression, or (c) susceptible to a specific disease regardless of exposure. In this scenario, (a) the worker could be monitored for markers of exposure and toxicity to a specific chemical, (b) the worker could be monitored for the development of an identified marker indicative of early disease, or (c) the worker could be evaluated for the presence of molecular events specific to the manifestation to the disease in question. Armed with this type of knowledge, one could take action to prevent or avoid subsequent exposure to the specific chemical or gain awareness and practice avoidance to all known environmental causes of the particular disease in question.Further, in situations of outward observable disease, many are tempted to ask the following question: “Is this disease due to my past exposures?” With advanced understanding of the relevance of molecular landscapes and of overlapping molecular events between an environmental carcinogen and a specific disease outcome, it may be possible to answer this question in a more definitive way. Caution must be taken, however, when interpreting these molecular events as we do not suggest that the WoE approach to assigning significance to an overlapping molecular landscape should be used as a surrogate for standard epidemiologic methods for determining causation. Rather, we view this method as evidentiary support for causal inference. Further research is needed to understand how molecular landscapes correlate with individual exposures to carcinogens, molecular mechanisms for disease progression, and disease etiology.The true usefulness of this approach is to further our understanding of molecular epidemiology on a population level. As the field of molecular epidemiology progresses quickly, biomarkers for exposure, disease progression, and disease outcome are becoming more and more prevalent. Given the increasing amount of molecular information that researchers have access to through the published literature, a system such as the WoE approach illustrated herein is a useful tool for sorting, categorizing, and prioritizing the most meaningful information. Using such a framework, researchers can determine ways to take advantage of best practices in identifying exposure scenarios and defining biomarker(s) relevant to the exposure-disease paradigm.


The receptor-ligand interaction evaluation is one important step in rational drug design. The databases that provide the structures of the ligands are growing on a daily basis. This makes it impossible to test all the ligands for a target receptor. Hence, a ligand selection before testing the ligands is needed. One possible approach is to evaluate a set of molecular descriptors. With the aim of describing the characteristics of promising compounds for a specific receptor we introduce a data warehouse-based infrastructure to mine molecular descriptors for virtual screening (VS). We performed experiments that consider as target the receptor HIV-1 protease and different compounds for this protein. A set of 9 molecular descriptors are taken as the predictive attributes and the free energy of binding is taken as a target attribute. By applying the J48 algorithm over the data we obtain decision tree models that achieved up to 84% of accuracy. The models indicate which molecular descriptors and their respective values are relevant to influence good FEB results. Using their rules we performed ligand selection on ZINC database. Our results show important reduction in ligands selection to be applied in VS experiments; for instance, the best selection model picked only 0.21% of the total amount of drug-like ligands. In these simulations a docking algorithm predicts the best position and conformation of a drug candidate  within the constraints of a target receptor binding site in order to correctly estimate their stability in terms of free energy of binding scores [One of the most important steps in rational drug design (RDD) is the receptor-ligand interaction evaluation at an atomic level, which is achieved through molecular docking simulations . This isg scores , 2. in silico. This selection can be performed with different approaches. For instance, it may make use of molecular coordinates or consider a set of molecular properties, also called molecular descriptors.In the early stages of the drug discovery process researchers can be interested not only in understanding the interaction between one receptor-ligand but also in testing a set of different drug candidates in a process defined as structured based virtual screening (VS) . This VSWith the aim to contribute to a more effective ligand selection, in this work we focus on a new strategy to easily and efficiently describe important characteristics that indicate promising compounds to be investigated in VS experiments. Initially we proposed data warehouse (DW) schemas which are able to integrate molecular descriptors from different databases and relate them with VS experimental data . With thTo validate our proposed infrastructure, we performed a VS case study using AutoDock4  which coOur approach to preprocess the input files is primordial to achieve the expected data mining results. Over these input files we applied the J48 decision tree algorithm achieving up to 75% of accuracy in experiment 1 and up to 84% in experiment 2. From the induced models of experiment 2 we extracted rules used to effectively select ligands from ZINC database. The rule that produced the smallest selection picked only 0.21% of the total amount of drug-like ligands in ZINC, while the rule that resulted in the largest selection picked 25% of this total. In face of this, our results show important reduction in ligand selection to be applied in VS experiments. Despite the stored data, our DW is sufficiently generic to store as many structures, molecular descriptors, and molecular docking experiments as needed. Likewise, our data mining methodology is generic enough to be applied over any data stored in the proposed DW.The remaining of this work is structured as follows. x, y). In this tuple x is a set of predictive attributes and y is the class label . The learning step in classification task builds a model f where each attribute in x is mapped to one of the predefined discrete-valued and unordered target attribute y [According to Tan et al.  classifiribute y , 9. TherDecision trees output is a flowchart-like tree structure in which the internal nodes denote a test on an attribute, each branch represents an outcome of the test and each leaf node is assigned a class label . Accordix for which the class label is unknown, the attribute values of x are tested against the decision tree and the path traced defined the prediction class [Decision trees can be used for classification since, given a tuple on class . In doinon class  classifion class ).Rational drug design  has been in silico technique where a set of large libraries of drug candidates are analyzed in order to identify those structures which are most likely to bind to a receptor target, typically a protein or an enzyme [Virtual screening (VS) is ann enzyme . The strTo perform the experiments of this work we had to choose some tools and databases. There are many molecular docking softwares, for example, FlexE , Gold 1, and AutBesides the molecular docking tool, the VS strategy involves the use of ligand databases. As reviewed in  the mostIn order to validate our proposed methodology we performed a case study with two experiments considering as the target receptor the protein HIV-1 protease (PDB Code: 1HPV) , which iIn the first case study (experiment 1) we have used 76 out of 100 ligands considered in  obtainedThe proposed infrastructure applied for mining molecular descriptors for virtual screening is depicted in In the virtual screening module we collect both data from ligands in public ligand databases and from proteins structures in a PDB format. In these ligands and protein we perform molecular docking simulations and hold the respective results. From the diverse ligand databases we collect the molecular descriptors for each ligand being used. All data related to virtual screening and ligand databases modules are properly processed and inserted into the DW we have developed . The stoIn databases repositories of ligands we can find different information about compounds and such information can be very relevant for virtual screening. A wide number of this type of repositories are available for different research purposes. Among them we can mention the Cambridge Structural Databases (CSD) , ChemBanAll these databases store a different set of ligands. All of them provide for the stored ligands both their spatial coordinates and a set of properties or molecular descriptors. These properties are encoded information from the molecular structures where the molecular descriptors become numerical values representing such information , 29.molecular weight (MwT);P);predicted octanol-water partition coefficient (lognumber of hydrogen bond donors (HBD);number of hydrogen bond acceptors (HBA);number of rotatable bonds (NRB);apolar desolvation energy (ADE);polar desolvation energy (PDE);total polar surface area (TPSA);charge (Ch).In  it is poData warehouse can be defined as a repository holding information from multiple sources . This inThe mentioned DW  containsIn order to validate our proposed methodology and DW we performed a case study considering the HIV-1 protease receptor and 76 ligands as we described on Protein stores only the information about the HIV-1 protease receptor;Mol descriptor, database, ligand, and atom save the information about the 76 used ligands (considered in experiment 1), comprising their structures, molecular descriptors, and provenance data;Mol descriptor, database, ligand, and atom save the information about the 410 used ligands (considered in experiment 2), comprising their structures, molecular descriptors, and provenance data;Experiments record the molecular docking simulation results.Following the previous steps the results were stored on DW tables:Thus, using the stored data in our DW we generated the appropriate data mining inputs. In our case study of experiments 1 and 2 our input considers as predictive attributes the values of the 9 most suitable molecular descriptors and as target attribute the value of FEB obtained from the molecular docking simulation between the receptor and a determined ligand. For experiment 2 our input considers as predictive attributes the same 9 molecular descriptors which are presented in In our case study we choose to apply the C4.5  classifiIn order to validate the new strategy proposed we performed two case studies: experiment 1 is applied to validate our architecture and experiment 2 is used to generate a set of interesting rules about the performed docking experiments considering as target receptor the protein HIV-1 protease (PDB Code: 1HPV) .F-measure (FM), a rate related to the precision, and recall where higher values mean better models. For experiment 2 we also analysed the induced decision trees using the Ordinal Classification (OC) Index metric [From the mining results with the J48 algorithm we evaluate the induced models by typical measures: the rate of correctly classified instances, accuracy , the size of the tree , the root mean-squared error (RMSE), and mean absolute error (MAE) . We also considered the x metric .As we mentioned before we induced 6 decision tree models using J48 algorithm with default parameters of execution: discretizing the FEB value in 2, 3, and 4 classes and considering the two discretization methods, by equal frequency (Method 1) and by equal width (Method 2) for each case. For discretization in 2 classes, we obtained the same results for Methods 1 and 2 since they induced classes that have about the same number of instances in the two cases. These were the best models with an accuracy of 75% and an interpretable final tree model. Observing the evaluation measures of the 3 classes' inputs, discretization of Method 2, by equal width, obtained better results than discretization by equal frequency. With respect to the 4 classes' inputs, we can see the worst accuracy values for both discretization methods.The first rule indicates that ligands having the molecular weight lower than 246 mole have chance to be promising if they have over 2 hydrogen bonds donors as well as present a positive charge.In the other hand, if we look for bigger ligands, that is, ligands with a molecular weight larger than 246 moles, it is necessary that they have a flexibility with less than 4 numbers of rotatable bounds and an apolar desolvation energy lower than 1.92 kcal/mol.The induced decision tree model with two classes is depicted in In this VS experiment we considered a new set of 410 ligands and the same HIV-1 protease as receptor. We also prepared the same 6 input files as detailed on In order to better analyse the induced decision trees we applied the metric Ordinal Classification (OC) Index . The OC F-measure, and OC are obtained when considering the discretization Method 2, by equal width. Between these results the best one is for two classes of FEB: Good and Bad as detailed on Analysing Tables Although the best results are obtained with 2 classes, it is also important to analyze the rules related to Excellent FEB values. Thus, we choose to extract rules form the decision tree obtained with the four classes and discretization Method 2 by equal width detailed on P, number of rotatable bonds (NRB), and charge. We can conclude that for this target receptor these molecular characteristics are directly related to Good and Excellent FEB values.Analysing the induced decision trees of experiment 2 and including the results with 3 classes we can see that the molecular descriptors that are more frequent on these trees are the molecular weight (MwT), logThe main objective of this work is to propose a methodology to select the most promising ligands for a target receptor. In order to achieve that we use molecular descriptors characteristics obtained from decision trees induced from molecular docking experiments results of a small set of ligands. Following our proposed methodology  the nexthttps://zinc.docking.org/browse/subsets/). The rules used by ZINC to generate this subset [First, to compare our selection results, we choose the drug-like subset from ZINC database with 15,798,630 compounds in the August of 2013  present in the plasmatic membrane [ in silico analyzed.We intend to further perform VS experiments considering more ligands and refine the generated rules. Besides, we are managing to apply this proposed methodology into the selection of new promising compounds to bemembrane . Membranmembrane . Thus it


The increased interstitial fluid pressure (IFP) from vasogenic edema, which results from a leaky blood-tumor-barrier (BTB), creates a barrier for transvascular drug delivery, particularly larger molecules that are unable to diffuse across and must resort to bulk fluid flow. We have previously used dynamic contrast enhanced-computed tomography (DCE-CT) to quantitatively evaluate the blood-brain-barrier (BBB) response in normal brain - and have shown that focused ultrasound (FUS) and microbubble (MB) treatment in a rat brain demonstrates a 3-4 times transient increase at our optimized parameters. The purpose of this study was to quantitatively evaluate the BTB response following FUS and MB treatment in rat glioma, in the context of improving drug delivery to brain tumors.At 10-15 days before treatment, 1 x 106 C6 glioma cells are surgically implanted using a stereotactic frame into a region in the right cerebral hemisphere of a Wistar rat. The rats were broken into 2 groups before treatment: 1) evaluating the acute response out to 4 h post for tumor and normal hemispheres (n=3), and 2) recovery at 2 h post followed by scans at 24 and 72 h post (n=5). The tumor is transcranially sonicated at previously optimized parameters that demonstrate transient opening in the normal BBB without any vascular or tissue damage - 10 ms burst length with 1 Hz repetition frequency for 120s, with an applied power of 0.5 W, using a 0.563-MHz FUS system. 2 ul/kg of Definity microbubbles are injected simultaneously with the start of the sonication. Axial CT scans as well as the DCE-CT perfusion maps were used to target the tumor. DCE-CT  was performed using a two phase protocol: 1st phase consisting of continuous 0.5 s rotations for 30 s, and a 2nd phase of 0.5 s rotations at 14.5 s intervals for 150s. 2.5 ml/kg of iodinated contrast agent was injected over 5 seconds simultaneous to the start of each DCE-CT scan. Proprietary CT Perfusion software was used to compute permeability surface area product (PS), cerebral blood flow (CBF), cerebral blood volume (CBV), and mean transit time (MTT) maps using a standard small molecule contrast agent (Isovue ~ 760 Da) that is able to diffuse across the BTB. The BTB PS was also evaluated using a much larger contrast agent  that mimics the delivery of agents such as monoclonal antibodies.Instead of a transient increase in BBB permeability that is seen in the normal brain, FUS and MB demonstrated a gradual decrease in BTB permeability in the hours following treatment (p < 0.05). An acute vascular shutdown, drop in CBF and CBV (p < 0.05), was also observed immediately following treatment, as previously reported in other tumor models. The drop in PS persisted at 24 h post (p < 0.05), returning at 72 h post, whereas the CBF and CBV returned back to baseline levels by 24 h post. A trend of increasing eXia penetration suggests that the decrease in BTB PS alleviates the elevated IFP that results from vasogenic edema and improves the penetration of larger drugs or molecules that are unable to diffuse across the BTB. When plotted against the MTT, which is the inverse of perfusion pressure, BTB PS (isovue) shows a moderate positive correlation (r2 = 0.71), whereas BTB PS (eXia) shows a weak negative correlation (r2 = - 0.50). Our findings indicate that although FUS and MB treatment at our parameters may not necessarily be useful for increasing the delivery of small molecule drugs, it may be more useful for increasing the delivery of larger molecules where the IFP is a barrier.


On the basis of theoretical calculations, we assign the observed peaks to the transition from the ground vibrational state, ν=0 to ν=9 and 10. Our method allows us to track single-molecular events, and it can be extended to work with any molecule by using normal mode frequency shifts to detect the dissociation. This survey spectroscopy serves as a bridge to the precision spectroscopy required for molecular ion control.Molecular ions can be held in a chain of laser-cooled atomic ions by sympathetic cooling. This system is ideal for performing high-precision molecular spectroscopy with applications in astrochemistry and fundamental physics. Here we show that this same system can be coupled with a broadband laser to discover new molecular transitions. We use three-ion chains of Ca Studying the spectra of molecules typically requires large samples, which can be difficult to achieve for hard-to-generate ions. Here, the authors obtain spectra from single CaH+ molecules in a three-ion Columbic crystal, observing new molecular transitions. It can be applied to study the vibrations of molecules from diatomics with a single electron16+ by two-photon resonant photodissociation of single-molecular ions sympathetically cooled by two Ca+ ions. The overtone is excited by a mode-locked laser in order to span the thermally occupied rotational states. A second photon dissociates the molecule and the resulting Ca+ is detected by a change in fluorescence. The change in the rate of dissociation as a function of the mode-locked laser wavelength reveals two peaks. We assign these peaks to the overtone transtions from the ground vibrational state ν=0 to ν=9 and 10 based on theoretical predictions. Our observation is the first measurement of a rovibrational transition in CaH+ and shows that single-molecular ion experiments can be used to discover new transitions.We present the observation of two vibrational overtones of CaH40Ca+ ions by isotope selective photoionization and then laser cooling the ions to form a three-ion chain. The 40Ca+ ions are trapped in a radio-frequency Paul trap (r0=0.6 mm) driven at Ω=2π × 14 MHz to confine the ions radially, whereas static DC voltage applied at the endcaps confines the ions axially. The trap is kept at a base pressure of ∼4 × 10−9 Pa. The ions are detected by laser-induced fluorescence at 397 nm onto a photon multiplier tube and electron multiplying charge coupled device camera. A narrow band-pass filter is used to ensure that only 397 nm light is detected. The experimental apparatus has previously been employed for sympathetic heating spectroscopyWe start with loading three 40CaH+ molecule is produced by leaking ∼5 × 10−7 Pa of molecular H2 into the chamber via a leak valve. The 40CaH+ is produced via reactive collisions in the gas phase between 40Ca+(4P1/2) and the H2 as 40Ca++H2→40CaH++H. The product molecule quickly relaxes to its electronic ground state X 1Σ+  nm compared with the theoretical value of 813.3 nm and ν′=9←ν=0 transition to be at 890(3) nm compared with the theoretical value of 883.3 nm. This disagreement is in line with observed differences between calculated and measured vibrational transition frequencies in other metal hydridesThe spectrum clearly shows two peaks that we identify as the PT2 ref. ; Fig. 3.+ is to reduce the rotational temperature by sympathetic cooling with neutral atoms, and then rotationally resolve these transitions and the fundamental transition24+–CaH+ crystalsOur experimental setup was intended for high-precision quantum logic spectroscopyOur current experiment takes advantage of the fluorescence of the dissociated product and can be easily applied to other fluorescent product ions. The method can be modified for dark product ions by periodically blocking the dissociation laser and measuring the mass of the product ion. This can be achieved by monitoring the sidebands of the atomic ions and calculating the mass of the unknown ion from the observed normal modes5+ .


EC 4.2.1.78) catalyzes the Pictet–Spengler condensation of dopamine and an aldehyde, forming a substituted (S)-tetrahydroisoquinoline, a pharmaceutically important moiety. This unique activity has led to NCS being used for both in vitro biocatalysis and in vivo recombinant metabolism. Future engineering of NCS activity to enable the synthesis of diverse tetrahydroisoquinolines is dependent on an understanding of the NCS mechanism and kinetics. We assess two proposed mechanisms for NCS activity: (a) one based on the holo X-ray crystal structure and (b) the ‘dopamine-first’ mechanism based on computational docking. Thalictrum flavum NCS variant activities support the dopamine-first mechanism. Suppression of the non-enzymatic background reaction reveals novel kinetic parameters for NCS, showing it to act with low catalytic efficiency. This kinetic behaviour can account for the ineffectiveness of recombinant NCS in in vivo systems, and also suggests NCS may have an in planta role as a metabolic gatekeeper. The amino acid substitution L76A, situated in the proposed aldehyde binding site, results in the alteration of the enzyme's aldehyde activity profile. This both verifies the dopamine-first mechanism and demonstrates the potential for the rational engineering of NCS activity.Norcoclaurine synthase (NCS) ( EC 4.2.1.78) is responsible for the formation of (S)-norcoclaurine via a Pictet–Spengler condensation of the tyrosine derivatives dopamine and 4-hydroxyphenylacetaldehyde (4-HPAA) -Norcoclaurine is the precursor to all benzylisoquinoline alkaloids (BIAs), a diverse group of ∼ 2500 natural products ,1816,18]cis-trans isomerization and the rotation of the entire aldehyde R-group. This step is vital for the subsequent rate-determining cyclization and deprotonation, which establish the chirality of the product. However, there is no evidence for this reorganization: the holo crystal structure could only provide evidence for the initial arrangement of the substrates.In this mechanism, the intermediate imine undergoes a rearrangement which includes an iminium Kb = −1.7) to deprotonate the 3-hydroxy of dopamine in the rate-determining step; a basic active site residue is required . Thi. ThiTfNC A) Fig. B and 4. A key factor in the biocatalytic utility of NCS is its ability to turn over a wide variety of structurally diverse aldehydes. Docking calculations provide a structural explanation for this observation: the most favourable binding modes for reaction intermediates show that only the aldehyde R-group is partially exposed to the solvent Fig. . The pooin silico docking results  , which is the result of burying a charge in a hydrophobic environment  and a change in charge position (E110D) eliminate activity supports the suggestion that Glu110 acts as a base, abstracting the quinone 8a-H Fig. B, step eKm values in the Y108F variant -THIQs. The dopamine-first mechanism predicts that Leu76 is proximal to the aldehyde R-group - and (R)-citronellal but reduces activity towards 4-HPAA and hexanal (Table S)-citronellal where the apparent kcat is doubled both with respect to dopamine and the aldehyde. Interestingly, apparent Km values are largely unaffected by the substitution. The data may also suggest that citronellal inhibits the enzyme at high concentrations - and (R)-citronellal intermediates are relatively close to Leu76: at 3.65 and 3.75 Å respectively. Removing the steric bulk close to this methyl moiety may enable greater conformational freedom in the intermediate, entropically reducing the reaction energy barrier. The enhancing effect of the amino acid substitution may be more pronounced for (S)- rather than (R)-citronellal simply as consequence of proximity of the methyl group to Leu76.Docking calculations of the imine-citronellal reaction intermediates suggest a molecular origin to this activity increase Fig. . The metThe substitution L76A has modified the aldehyde activity profile of NCS. This supports the dopamine-first reaction mechanism and also demonstrates the potential for engineering NCS towards other substrates and activities.TfNCS variants appear to support the dopamine-first mechanism. By avoiding the use of phosphate buffer in enzyme assays, we observed novel kinetic parameters that show NCS operates with low catalytic efficiency. This can account for its apparent ineffectiveness in recombinant in vivo systems, and may indicate a possible in planta role as a BIA gatekeeper enzyme. The amino acid substitution L76A, in the proposed dopamine-first aldehyde binding site, resulted in a change in the aldehyde activity profile, strongly supporting the mechanism in question.In the present study, we have described and assessed two different mechanisms of NCS activity: the HPAA-first mechanism and the dopamine-first mechanism. Activities of in vitro and in vivo. For example, double-reciprocal plots with an appropriate inhibitor will provide a kinetic assessment of the compulsory-order binary mechanism proposed in the present study. It may also be possible to monitor the interaction between NCS and substrates via direct methods such as NMR . Do. DoTfNCSess = 8) . Unless propka, version 3.1 /MeCN from 9 : 1 to 3 : 7 over 6 min, at a flow rate of 1 mL·min−1. The column temperature was 30 °C, and compounds were detection by monitoring A280. Retention times were 4.9 min and 5.9 min for (S)-norcoclaurine and the hexanal product, respectively. The enantiomeric excess (ee) was determined by chiral HPLC analysis using a Supelco Astec Chirobiotic T2 (250 × 4.6 mm) column  and an isocratic MeOH  mobile phase at 1 mL·min−1 and 30 °C. Compounds were detected by monitoring A230.Analyses were performed on a HPLC system consisting of an LC Packing FAMOS Autosampler, a P680 HPLC Pump, a TCC-100 Column oven and a UVD170U Ultraviolet detector . Quantification of enzyme substrates and products were determined by analytical reverse-phase HPLC with a C18 (150 × 4.6 mm) column  and a gradient of HS)- and (R)-citronellal were purchased from Sigma-Aldrich. 4-HPAA and racemic BIAs for HPLC standards were synthesized as described by Pesnot et al. , 1.87 , 2.01 , 2.86–3.00 , 3.29–3.34 , 3.50 , 4.34 , 6.60 , 6.64 ; 13C NMR  δ 14.3, 23.5, 25.7, 26.1, 32.7, 35.1, 41.0, 56.7, 113.9, 116.2, 118.3 , 123.7, 124.3, 145.9, 146.7, 163.2 ; m/z [HRMS ES+] found MH+ 236.1639. C14H22NO2 requires 236.1651.Hexanal, dopamine hydrochloride, (t et al. . Charact


It is currently unclear whether an adolescent with 47,XXY Klinefelter syndrome will be better off having testicular sperm extraction (TESE) performed in an effort to ‘preserve fertility’ for the future or, alternatively, should be advised to simply wait until adulthood when he and his partner are ready to begin a family. This report will provide data suggesting that there is no obvious ‘preservation’ benefit and that recommending TESE to the 47,XXY boy and his parents may not be as helpful as it might appear and may be overly aggressive. When testicular sperm extraction (TESE) became a standard technique for the discovery and retrieval of sperm in the NOA patient, it was subsequently successfully applied to KS adults, who had previously been considered sterile. Published reports from many different centers describe rates of sperm retrieval in these 47,XXY adults to be approximately 50–70%, along with excellent pregnancy rates and healthy 46,XY or 46,XX offspring, as recently reviewed by Majzoubet al.2.Klinefelter syndrome (KS)  is the most common chromosomal disorder in men, affecting one in 600 newborn males, and is etiologic in up to 11% of non-obstructive azoospermic (NOA) males7. Based upon the knowledge that not all adult KS males will have spermatozoa harvested upon TESE, several clinical investigators have pursued a course of action to determine if TESE should be offered and applied to the 47,XXY adolescent in order to “preserve fertility potential”9. This submission will address this vexing question from many different angles and try to answer whether this is an overly aggressive approach with more harm than benefit or if this is an appropriate strategy with more benefit than harm.For a variety of reasons, KS may occasionally be diagnosed prenatally, in infants, in young boys, or in adolescent males10. More recently, Aksglaedeet al. reviewed 166 non-mosaic KS boys and demonstrated that there is no rapid, inexorable decline in their serum testosterone values  spanning early adolescence to adulthood11.The underlying tenets for recommending TESE in adolescent KS males is that fertility potential and the presence of testicular spermatozoa are maximal at puberty or just after, and that both are precipitously and irrevocably lost over the next several years. Is that actually true or has fiction become belief that has become fact that has become dogma? Additionally, some feel that testosterone levels plummet after puberty and require replacement, and TESE should be carried out prior to that. However, in 1985, Saltenblatt concluded that testosterone levels settle at low-normal values for most with KS after puberty and remain at those levels into adulthood14. Even after invading and populating the emergent seminiferous tubules, numerical increase continues until birth. With the onset of the mini-puberty  during the first few months of life, proliferation and differentiation (of some) to type A dark (Ad) spermatogonia commences and, when this temporally limited hypothalamic-pituitary stimulation stops, the spermatogonia become quiescent until puberty, although there may be a gradual diminution in the absolute numbers of spermatogonia in the first year of life16. The vast majority of these resting cells would be 47,XXY but, occasionally, it is thought the supernumerary X chromosome is lost during an earlier mitotic replication and the resultant spermatogonial stem cell or type Ad spermatogonia is, therefore, normally diploid 17. These are thought by many to be the cells that eventually, upon initiation of puberty, will be capable of completing the full process of spermatogenic differentiation , their progeny being fully functional haploid spermatozoa. However, the more numerous, by orders of magnitude, 47,XXY spermatogonia suffer a meiotic block, arrest, and become apoptotic18. Whether it is simply the trisomic state or the overexpression of X-linked, testis-expressed genes that lead to this demise is unclear, although the latter hypothesis has more evidence behind it20. The legacy of this self-destruction is wide swaths of the testicular parenchyma with seminiferous tubules that are empty ghosts or unrecognizable and fibrotic. By happenstance, every once in a while, a seminiferous tubule in which a 46,XY spermatogonium found itself at home survives, incubates, and cultivates the normal machinery responsible for complete spermatogenesis21. So, if 47,XXY spermatogonia have no ability to birth whole spermatozoa, is there a reason to harvest and cryopreserve them before they become apoptotic early in puberty? The answer would appear to be “no”, as concluded by Oates22. Indeed, with regard to fertility preservation in even younger males with KS, as Gieset al. cautiously posit, “given these controversies, banking testicular tissue from prepubertal KS boys should be performed only in a research framework”23, even though parents of KS boys would be in favor of it24.At conception, the nascent embryo is 47,XXY and all cells, including those destined to become gonocytes and eventually spermatogonial stem cells, will have this same chromosomal constitution. As the spermatogonial stem cell precursors and stem cells themselves migrate to the gonadal ridge from the yolk sac, they expand mitotically in number and begin the slow process of differentiationGiven the above discussion that spermatozoa arise from random 46,XY spermatogonia scattered about a sea of fibrotic tubules, the next obvious question in the search to answer whether it is advantageous to perform TESE in an adolescent KS male as opposed to waiting until adulthood would naturally be whether the chance of finding sperm upon TESE is greater in the adolescent than it is in the adult. If so, that would be a rationale for TESE in these younger males, but if not there would seem to be no benefit in doing so. That is, is there a great likelihood that these competent and capable 46,XY populated seminiferous tubules will also disappear during puberty or in the next several years, perhaps collateral damage of the near total annihilation of the neighboring 47,XXY spermatogonia and their home tubules and, thusly, they and any sperm they produce should be harvested and saved for the future as soon as possible?31. Retrieved spermatozoa could even be intentionally frozen prior to use in conjunction with ICSI, as first demonstrated by Oateset al., and this is now routine clinical practice32. In their recent systematic review and meta-analysis, Bernieet al. concluded that the sperm retrieval rate (SRR) was higher with micro-TESE (involving the use of an operating microscope) than with conventional TESE33 and approximates 50–60%, depending upon the center and the surgeon. Tournayeet al. initially pioneered its application in adult KS men, and the first pregnancy was documented by Palermoet al.35. Numerous subsequent publications confirmed this initial proof of concept and added to the growing cumulative statistic of approximately 50–60% likelihood of sperm retrieval in this subpopulation of NOA men2.Sperm seen within the ejaculate are probably only a small percentage of the total number produced in the testis when viewing the entirety of the testis parenchyma as a single manufacturing unit. When that output falls below a certain minimum number, but not to zero, no sperm can be found downstream in the seminal fluid, but some of the relatively few that have been created may still be identified when combing through and dissecting that testis tissue factory itself. Shortly following the introduction of intracytoplasmic sperm injection (ICSI), it was realized that testicular sperm was capable of fertilization, embryo development, and pregnancy. TESE quickly became the standard therapy offered to men with NOA of all types in an effort to find and use individual spermatozoa to achieve male genetic parenthoodet al. first reported in 2001 the use of TESE in an adolescent male in order to ‘preserve’ his fertility before he was started on spermatogenically suppressive testosterone replacement36. The authors carefully presented their data and the decisions that led the patient and his parents to choose this approach in a cautionary tone, advocating only that TESE in the adolescent KS male needed more study and consideration before becoming viewed as routine, always beneficial, and standard. Building upon that idea, a handful of subsequent reports presented data on testis biopsy or TESE in very young/adolescent KS boys with varying results. In 2004, Wikstromet al. reported that they were not able to find any spermatozoa in the histological preparations of single-site biopsies on 14 boys aged 10–14 years37. They noted a minimum number of type A pale (Ap) and Ad spermatogonia in seven of the 14, all within the age range of 10–12.5 years, but no spermatogonia of any kind in the remaining seven who had an age range of 11.7–14 years. In 2012, Gies and colleagues, who also performed a single-site biopsy but of “large volume”, reported that they too could not document full spermatogenesis on histological analysis in seven KS boys, aged 10.2–15.6 years38. Spermatogonia were seen in four of the seven in the upper age range of their group (13.3–15.9). Another seemingly disappointing report by Rivaset al. in 2013 stated that only one KS adolescent out of five operated upon had spermatozoa visualized, even though they carried out single-site bilateral TESE39. Mehtaet al., also in 2013, using a microsurgical TESE approach reported positive news with sperm discovery in seven of 10 adolescents, ages ranging from 14–22 years: an SRR of 70%40. Plottenet al. in 2015 described an SRR of 52% in “young” KS patients, aged 15–239. Finally, Nahataet al. reported a 50% SRR in 10 adolescent KS boys aged 15–23 when performing microTESE41. Sperm was identified in five KS boys  but was not discovered in the remaining five KS boys , indicating no real pattern or prediction based upon age at the time of TESE.Damaniet al. performed exactly that study showing prospectively, as detailed previously, an SRR of 52% in their “young” group and 62%  in their “adult” group, aged over 23 years9. At Weill Cornell Medical College, Dabaja and Schlegel confined their reported results to 127 adult KS men and showed an SRR of 65%, which is similar to that of 70% (7/10) reported by Mehtaet al. at the same institution42. As stated and suggested by Nahataet al. in their conclusion, “…there is no clear benefit to performing TESE/unilateral microTESE in adolescence…”41. In total, then, although perhaps intuitively satisfying, sensible, and gratifying, SRRs in the small numbers of adolescent KS boys subjected to TESE appear to be the same as those SRRs in adult KS men subjected to TESE. In general, the sperm retrieved from the adult is used immediately as the source of sperm for ICSI and not cryopreserved. Will cryopreserved spermatozoa or testis tissue be equally as useable and successful after years of storage as would necessarily be the case if harvested from a young boy not ready to conceive for years? Future reports detailing the success of using this sperm (obtained many years previously) will be necessary to answer this question. This will include those adolescents who have had microTESE prior to the institution of spermatogenically suppressive exogenous testosterone therapy.As above, SRR rates of TESE in KS adults approximate 50–60%. But are there data to compare within the same program, adolescent to adult? Plotten43. Although these conclusions have been made based on a limited data set from Okadaet al.43 , this is by no means certain. As a matter of fact, the data from the Cornell group44 show that the SRR in KS men was 71% in the 22–30 year age group, 86% in the 31–35 year age group, and 50% in the 36–50 year age group. The group age intervals are not of the same time spans, and the SRR is higher when you are 33 than when you are 26  and is still 50% when greater than age 36. As they state, “In addition, ROC analysis of these data yielded an AUC of 0.58, indicating that there is no best age to predict SRR based on our data”. So it appears that even in the publications discussing whether or not TESE rates are less in the ‘older’ adult than in the ‘younger’ adult (aged 35), there is still a rate of success that is close to, and not much better than, that claimed for adolescents.This is another way of asking the same question: should we be performing TESE in the adolescent to preserve fertility that will be irrevocably lost as the years pass and we do nothing? This notion also arises from very limited data suggesting that sperm production, as determined by results of TESE (namely SRR), declines after a certain age (35 or so) and as such it might be best to harvest tissue as soon as the diagnosis is made, whatever the ageFinally, when comparing overall adolescent rates to overall adult rates of TESE success, and although difficult to prove, there would be little reason to believe that sperm production would magically begin years after the onset of puberty, e.g. age 16 or 17. Therefore, since 50 out of every 100 KS boys have sperm recovered in their testis tissue and 50 out of every 100 KS adults have sperm recovered from their testis tissue, and if a decline in sperm production occurred during this time frame to zero values of, for argument’s sake, 10%, then 10% of maturing KS males (adolescent to adult ages) would have to begin to produce sperm to keep the two groups equivalent, as far as SRR rates go. Is it likely that 10 (or whatever absolute number is chosen) males have a precipitous and total decline in sperm production as they advance from adolescence into adulthood and, to keep the rates of what we actually find upon TESE balanced, 10 males begin to suddenly produce sperm as they advance from adolescence into adulthood? Or is it more likely that those who have seminiferous tubules populated by 46,XY spermatogonia capable of undergoing the full and complete sequence of spermatogenesis maintain those same tubules throughout the years of adolescence and on into adulthood? It may be the exact same individuals who have spermatozoa found in adolescence that are those found in adulthood. Where would the benefit be? Harm could certainly come to the adolescent and his parents when no sperm are retrieved—a potential heavy burden to carry in the difficult teenage years.in vitro cycle for her is what they would like to do in an effort to build a family. Since the incidence of KS syndrome is 1:600, practitioners of all types are going to have many KS males as their patients, will be involved in this debate and controversy, and will need to be aware of the issues involved. This list includes, but is not limited to, obstetricians (amniocentesis results), pediatricians , endocrinologists (detected during exploration for hypogonadism and failure to initiate puberty in the most severely affected), urologists, and reproductive endocrinologists (elucidated at the time of an infertility work-up). As it usually is, the final answer will probably lie somewhere in the middle and be determined on an individual patient-specific basis.The basic biology of spermatogenesis and how it is altered in the KS testis is important to appreciate when trying to formulate appropriate clinical pathways vis-à-vis fertility and biological paternity. Although it seems obvious to some that KS boys benefit or even require testis tissue surgery and freezing of any sperm found as soon as they are identified in the adolescent years, the data do not support the temporal necessity to do so. It may be just as reasonable, if not more so, to perform that same surgery at a later date, in adulthood when the patient and his partner can make an informed choice as to whether an operation on him and an


Few details endure about the life and family circumstances of Flemish painter Cornelius Norbertus Gijsbrechts. We know that he was born in Antwerp, Belgium, and his talent earned him a position of court painter with two Danish kings, Frederick III (1609−1675) and Christian V (1646−1699). In 1660, Gijsbrechts was accorded membership in the prominent, influential Guild of St. Luke. Over his career, the artist worked in various locations, including Antwerp, Regensburg, Hamburg, and Copenhagen, and it was in the latter city where he created his signature series of trompe l’oeil (deception of the eye) paintings during 1668−1672.Trompe l’oeil, a subgenre of still life painting, embraces use of realistic imagery to create an optical illusion of depth in works that can be perceived as real, three-dimensional objects rather than flat paintings. It flourished from the Renaissance onward, though murals of trompe l'oeil art are found among the ruins of Pompeii and Herculaneum and remain popular today. According to the National Gallery of Art, “The discovery of perspective in fifteenth-century Italy and advancements in the science of optics in the seventeenth-century Netherlands enabled artists to render objects and spaces with eye-fooling exactitude.”Trompe l’oeil with Studio Wall and Vanitas Still Life is a prime example of this inventive subterfuge and also of a vanitas painting, a meditation on mortality. Gijsbrechts makes a skull the focal point of the painting, ensuring no one misses his allusion to the temporal nature of life. Other symbols supports the theme of transience: smoke trailing from an extinguished candle and an hourglass tipped on its side symbolize death; a violin, tankard, clay pipe, and tobacco represent fleeting pleasures. The unity of these symbols represent the ebb and flow of birth, death, and resurrection, encouraging both a wistful examination of life’s purpose and a solemn acceptance of death’s finality.This month’s cover painting Gijsbrechts pulls the scene back and reveals the vanitas on a temporary canvas hung on a wooden wall, surrounded by the tools of his craft, including brushes and a palette featuring the colors he used in his construction of the painting. A miniature self-portrait affixed to the frame reminds viewers that art endures beyond the life of the artist. Those who fall for the deception and perceive the vanitas as the sole painting miss the message that Gijsbrechts intends: nothing is what it seems without context.In trompe l’oeil, the background imagery surrounding the painting’s center provides perspective and clues that engage viewers and contribute to understanding the overall work. Disease surveillance for emerging infections relies on a somewhat similar, albeit more complicated process. To gain perspective, one must assess a complex but incomplete set of intertwined, dynamic factors, which may include antimicrobial drug resistance, climate change, food production practices, global mobility, the route(s) of transmission of the etiologic agents, and ecology of the pathogens.By their nature, surveillance data are representative of disease in populations, and they are always incomplete and sometimes inaccurate. It takes a critical, well-prepared mind to properly interpret and analyze the meaning and significance of surveillance data or unravel clues about how an emerging infectious disease spreads. Gijsbrechts’ portrait displays the tools and medium that he used to construct his illusion, but not everyone who views the painting will come to this realization. The experience of viewing a trompe l’oeil reminds us that seeing beyond our initial assumptions—whether we are studying art, investigating the outbreak of an emerging infection, or analyzing reams of data—requires that we connect background information, delve beyond the surface, and recognize patterns and aberrations.


Arthrospira  was the single source for nanofiber biofunctionalization. We investigated nanofibers using PHB extracted from Spirulina and the bacteria Cupriavidus necator and compared the nanofibers to those made from commercially available PHB and PHB-HV. Our study assessed nanofiber formation and their selected thermal, mechanical, and optical properties. We found that nanofibers produced from Spirulina PHB and biofunctionalized with Spirulina biomass exhibited properties which were equal to or better than nanofibers made with commercially available PHB or PHB-HV. Our methodology is highly promising for nanofiber production and biofunctionalization and can be used in many industrial and life science applications.Electrospun nanofibers composed of polymers have been extensively researched because of their scientific and technical applications. Commercially available polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-valerate (PHB-HV) copolymers are good choices for such nanofibers. We used a highly integrated method, by adjusting the properties of the spinning solutions, where the cyanophyte  In recent years several innovative nanofiber technologies are being studied, such as the development of structural nanocomposites providing dual drug release through a combination of electrospinning and electrospraying  and the Polymer nanofibers produced by electrospinning are currently receiving a great deal of interest, largely because of their technical and life science applications. These highly porous, nonwoven fibers have a large surface area and can be used for diverse applications, including the tissue engineering –10. For  in vivo and an appropriate time is extremely important for proper regeneration [The degradability is a parameter closely related to the solubility of the molds. The degradability is associated with the stability of the biomaterialneration . The mosneration .Nanofibers can also be biofunctionalized; that is they can be altered for specific functions and thus improved, by, for example, incorporating compounds such as antioxidants, anti-inflammatory, antibacterial, antifungal, vitamins, or other compounds . HoweverPolyhydroxybutyrate (PHB) and its copolymer with hydroxyvalerate (HV), poly (PHB-HV), are accumulated by some microorganisms where they act as intracellular storage compounds which can be used as a source of carbon and energy , 16. Com Arthrospira, usually Arthrospira platensis and Arthrospira maxima, have for many years been commercially available under the name “Spirulina”, which has been reported to be not only therapeutically effective in reducing cholesterol levels but also displaying antimutagenic and antiviral activity, potentially inhibiting HIV replication [Microalgae and cyanophytes are photosynthetic microorganisms that can double their biomass in 24 hours and not only produce PHB polymers but also biologically active compounds such as proteins, polyunsaturated fatty acids, vitamins, pigments, antioxidants. Cyanophytes from the genuslication  and tumolication . Spirulina biomass. Commercially available PHB-systems were also studied for comparison.We used PHB extracted from different types of microorganisms to develop nanofibers and used electrospinning to produce uniform nanofibers, which were then biofunctionalized by the incorporation of Spirulina platensis  and Cupriavidus necator bacterial PHB samples RE and CN ; commercially available PHB , and PHB containing poly  and 12% (w/v)  were also used for comparison. Nanofibers were electrospun from chloroform solutions of PHB and PHB-HV, with the properties of the spinning solutions and the resulting nanofibers being modified by adding from 0.2% to 2.2% (w/w) sodium chloride and 5% to 35% (w/w) Spirulina biomass , vacuum packed using a model Supervac400 vacuum packing machine , and stored in the dark at 4°C. The amount of ash, lipid, moisture, and protein in the biomass was measured using standard methods [Biofunctionalization  was acco biomass  cultivat biomass . The Spi methods .μL min−1 to 6.5 μL min−1. The capillaries had a positive electrode at their tip and a grounded aluminum collector between 150 mm and 200 mm from the tip and an applied electric potential ranging from 12.4 kV to 31.3 kV. To ascertain the best conditions for producing uniform nanofibers, the PHB and PHB-HV samples were tested at different concentrations, voltages, flow rates, distances from the capillary to the collector, and capillary diameters (data not shown).Electrospinning was carried out at 21°C by injecting the PHB solutions  through P ≤ 0.05). The mechanical properties of the nanofibers were evaluated using sets of parallelly orientated nanofibers spun on an electrode rotating. Stress-strain experiments were done by assessing elasticity, tensile strength, and breaking elongation using a BT1-FR0.5TN.D14 mechanical analyzer , with mean values being subjected to ANOVA and the Tukey test (P ≤ 0.05). Molecular weight of the polymers was determined by gel permeation chromatography (GPC) on a model SL1000 HPLC  equipped with a Polymer Standard Services column and two Knauer detectors (K2500 UV for detector 1 and RI for detector 2) and using hexafluoroisopropanol  as the mobile phase solvent at a flow rate of 0.5 mL min−1 and 23°C with a 100 μL sample injection volume, with molecular weight standard being 319,000 Da polystyrene . The thermal degradation and degree of impurity of the polymers were measured using a model SDTA851 thermogravimetric analyzer (TGA)  and approximately 10 mg of sample heated from 25°C to 700°C at 10°C min−1 in air with nitrogen, with the initial and highest degradation temperatures being determined from the first derivative of the TGA curves and the degree of impurities being characterized from the amount of sample remaining as ash at the end of the process [χc) of the polymers was calculated from the enthalpy of crystallization (ΔHc) and the enthalpy of melting (ΔHm) by differential scanning calorimetry (DSC) using a model DSC821e calorimeter  and 10 mg polymer samples sealed in aluminum capsules and heated and cooled between 25°C and 200°C at a rate of 10°C min−1, with the enthalpy of melting 100% crystalline PHB and PHB-HV (142 J g−1) being used as a standard [The apparent viscosity of the spinning solutions was determined using a model PK100 viscometer  and the conductivity of the solutions with a digital conductivimeter , respectively. Nanofiber mean diameter was calculated using a JSM-7500F scanning electron microscope  to measure 30 different points across SEM images of the nanofibers and mean nanofiber diameters being subjected to analysis of variance (ANOVA) and the Tukey test  and the 1200 nm . It has  1200 nm . HoweverRegarding thermal properties, the PHB-HV12 nanofiber sample had the lowest melting temperature, while Spirulina platensis PHB had lower initial and final decomposition temperatures than that seen for commercial PHB . Final d Spirulina and the commercial samples B showed the lowest levels of impurity at 1.3%, while C. necator sample RE showed the highest at about 4% w/w  for the PHB and the copolymer PHB-HV nanofiber samples used in our study are shown in is value . The addlina PHB . Nanofib present . Nanofib samples . Additio of 2.3% . The gen Spirulina biomass can provide additional optical functionalization of the nanofibers and affect the transmission of light because addition of biomass produces a nanofiber with a strong green color  in samples with added microalgal biomass, as well as enhanced elasticity of about 250.0 MPa, a tensile strength of about (8.0 MPa), a breaking elongation of about 7.0%, and minor impurities (1.3%). The reason for the deterioration of the mechanical properties of the nanofibers biofunctionalized with Spirulina biomass was probably due to biomass aggregation resulting in a heterogeneous distribution of biomass in the fibers. The properties of the nanofibers made from Spirulina PHB were highly favorable when compared to nanofibers produced from bacterial and commercial PHB and PBH-HV. The methodology described in this paper can be adapted to develop nanofibers that can be used in tissue engineering. Further studies should aim at reducing nanofiber diameter even more so that it approaches the ideal Knudsen number of kn ≥ 1 and to enhance the mechanical properties of biomass-containing nanofibers.Some highly innovative life science applications that have been recently developed, such as tissue engineering, have created a demand for nanofibers composed of biocompatible, biodegradable polymers on a technical scale. The study evaluated nanofiber formation, thermal and mechanical properties, and selected optical properties and compared them with PHB systems from other sources. The conclusion is that the integrated approach taken here is highly promising in terms of nanofiber production, biofunctionalization, and life science applications. Nanofibers produced from PHB extracted from


The fluorescent lifetimes of the BODIPY–BODIPY dyad appear to correlate with the viscosity of the media, thus making this rotor a suitable small molecule viscometer. A symmetrical BODIPY–BODIPY dyad with a diyne linker was prepared in two steps; the lifetime decay of this rotor appeared to correlate with the viscosity of the media, thus making this dyad a suitable small molecule viscometer for molecular solvents. The potential of using the rotor to probe the viscosity of ionic liquids was also investigated. Importantly, the photophysical properties of these dyes can be tuned 4Viscosity is a fundamental property of fluid media, and therefore evaluating the viscosity in various environments using molecular rotors is an attractive area of modern research with numerous applications in chemistry, biology and drug discovery. Arguably, due to the sensitivity of fluorescence, fluorescent lifetimes as well as the potential for high-throughput screening and imaging, the use of fluorescent probes as molecular viscometers, might provide ample opportunities and advances.meso-position of the BODIPY scaffold, and no substituents are present in the 1- and 7-positions to allow for a rotation around the single C–C bond (A).5 In general, an increase in viscosity should suppress the rotation of the substituents around the BODIPY core in the exited state, and as a result suppress the non-radiative decay, which will lead to an increase in emission intensity, quantum yield, and lifetime.11 It could be assumed that the photophysical properties of BODIPY dyes, lifetimes in particular, are insensitive to variations in the polarity and pH of the media, thus making these dyes viable for monitoring viscosity changes within different types of media. Specifically, the lifetimes of BODIPY viscometers  showed a linear correlation with the viscosity in the 15–1000 mPa s range. The main application of these rotors has been the evaluation of viscosity of molecular solvents as well as cellular environments.11 Several examples for using BODIPY-based rotors as fluorescent pressure sensors have also been reported.13Several BODIPY-based viscometers have been recently reported. Typically, the rotor-moiety is attached at the C–C bond , A.5 In N-isopropylacrylamide-4-chloromethylstyrene) polymers were recorded by covalently attaching a BODIPY fluorophore to the polymer's side chain.14 Also, an effect of the so-called remote substituents, i.e., aromatic (phenyl and tolyl) moieties in the 3- and 5-positions of the BODIPY motif (B) on the ability to respond to changes in the media's viscosity was evaluated in a series of homologous, straight chain alcohols with increasing viscosity (ca. 0.5–10 mPa s).15Additionally, temperature-dependent changes in the microviscosity of poly, exhibited a linear correlation with the viscosity of THF–ethylene glycol mixtures.16 Concurrently, the fluorescence lifetimes increased from 2.3 ns to 3.7 ns as the concentration of ethylene glycol increased from 10 to 90%. Furthermore, a triphenylphosphonium-containing coumarin-BODIPY viscometer (D) was shown to report changes in viscosity within mitochondria.17 Recently, a chimeric BODIPY-Nile Red probe that is capable of reporting on both polarity and viscosity of the specific media was also developed.18Only a few examples of BODIPY dyads as molecular viscometers have been reported. Specifically, the fluorescence intensity as well as quantum yield of a chimeric viscometer, featuring a BODIPY–ferrocene motif , C, exhiscometer , D was s1) and alkyne-BODIPY (2) were prepared according to either a conventional synthesis19 or by a recently developed mechanochemical procedure,20 and the subsequent dimerization of dye 2 was accomplished under Glaiser-type conditions22 to furnish BD in a moderate yield  to τint = 3.060 ns in glycerol (η = 1457 mPa s). In the case of BD, the change was even more pronounced with τint = 0.326 ns in ethanol as compared to τint = 4.500 ns in glycerol. Although dyes 1 showed only a marginal (ca. 3-fold) increase in the fluorescent lifetime within the given viscosity range , i.e., 13.8-fold for BD as compared to 5.6-fold for 2, and 3.3-fold for 1, we chose to further explore the possibility of using BD as a potential molecular viscometer.The fluorescence spectra of dyes  Table S1, this chBD was studied in ethanol, propylene glycol, and glycerol , and 1,3-dialkylmidazolium ILs are among the most widely used and studied ILs.23 These solvents have found numerous applications, and they could be viewed as customizable materials since their physical properties, including viscosity, can be tuned via structural modifications of the cationic and anionic counterparts.26 The viscosity of the 1-alkyl-3-methylimidazolium ILs can be modulated over a wide range by simply adjusting the length of the alkyl chains, as well as the identity of the anion.31Next, in order to explore other types of environments, we decided to probe the potential of 37 This heterogeneity was also demonstrated to be crucial in controlling the outcome of several organic reactions performed in ILs.39Importantly, on the structural level, neat ILs could be viewed as heterogeneous, supramolecular polymer-like assemblies (or nanostructured domains) as opposed to molecular solvents that are homogeneous fluids.BD in several ILs, namely [C4-mim]PF4, [C4-mim]NTf2, and [C12-mim]NTf2 (3- and BF4-containing ILs). This was expected to facilitate the handling and manipulation of the ILs during the measurements.Here, we investigated the behaviour of mim]NTf2 . These Imim]NTf2 , which cmim]NTf2 , and , a linear correlation for all solvents was observed .Alternatively, it could also be proposed that unique behaviour of observed , which cBD was prepared in two steps from commercially available starting materials, and it appeared to be a viable molecular viscometer for molecular solvents. The assessment of the viscosity of ILs using BD was less straightforward, most likely due to specific-solvent interactions and/or the unique aggregation state (or heterogeneity) of specific ILs. Nonetheless, BD appears to be a viable viscometer for monitoring viscosity changes of molecular solvents; based on our recent study, BD might also be suitable for probing viscosity of cellular and membrane-like environments.41


This editorial is an annotation on the exciting research topic “Microbiotechnology based surfactants and their applications” that covers a compilation of original research articles, reviews and mini-reviews submitted by researchers enthusiastically working in the field of biosurfactants.Pseudomonas sp. IMP67 is effected by the ratio of monorhamnolipid (MRL) and dirhamnolipid (DRL) congeners. The MRL and DRL congeners were analyzed by thin layer chromatography and rhamnose quantification. Rhamnolipids from Pseudomonas sp. IMP67 also reduced the minimum inhibitory concentrations (MICs) of some antibiotics signifying the synergistic role of these rhamnolipids with antibiotics.Biosurfactants, which for a long time have been confused with bioemulsifiers, derived their name from biologically produced surfactants. The term “Surfactants” was, however coined by Antara products in 1950—which covered all products having surface activity, including wetting agents, emulsifiers, dispersants, detergents, and foaming agents. The terms biosurfactants and bioemulsifiers have been used interchangeably for a long time until a demarcation has been suggested by several researchers including  than the complex microbial marine community-consortia (20 mg/l)  as a carbon source which was higher than obtained in presence of DBT-sulfone (0.5 g/l) and the inorganic sulfate (0.44 g/l)  in presence of heavy oil fraction of crude oil as substrate. Their data particularly emphasized on Aedes aegupti mosquitoes. Wang et al. (Dietzia and provided insights into the biosurfactant production, oil degradation and removal potential of Dietzia maris As-13-3.Some other interesting studies that focus on rhamnolipids and their applications are also included under this special research topic. Madsen et al. , compareg et al. , for theFrom a simple idea of growing bacteria and fungi on immiscible substrates and producing surface-active compounds, to a hurl of more than 250 patents filed in close to three decades followed by a market value expected to reach $2,210.5 million by 2018, biosurfactant industry certainly stands on a substantial fundament. Such stimulating facts and figures are broadly discussed in the opinion article by Sekhon Randhawa and Rahman .Apart from their industrially diverse applications in the field of bioremediation, enhanced oil recovery, cosmetic, food, and medical industries biosurfactants can boast off their unique eco-friendly nature to attract consumers and give the chemical surfactants a tough competition in the global market. The pharmaceutical applications such as biological usage as antiviral, antitumor, antibiotic agents, as insecticides, fungicides, and immune-modulators or enzyme inhibitors have not been fully realized. With the stringent governmental regulations coming into effect in favor of production and usage of the bio-based surfactants, more and more companies are working on the commercialization of the production technology of biosurfactants and to bring down their higher prices. There is no dearth of astonishing applications of biosurfactants; the only challenge is their supply through bio-based production methods to meet the demands well in time.PR initiated the research topic and co-ordinated the entire editorial process. There are 11 manuscripts accepted for publication in this research topic contributed by 55 authors from UK, Denmark, Greece, Germany, South Africa, India, Brazil, Bahrain, Portugal, and China. He has initiated peer review process by inviting experts from Germany, Spain, USA, Trinidad and Tobago, India, Denmark, China, Bahrain, Malaysia, Japan, and UK. The reviewers' co-operation and timely responses to complete the research topic is highly commendable. KS was not assigned as Topic Editor for this research topic but she has contributed as Co-author in this Editorial commentary. She is an expert on this topic and has been working specifically in this field, she has her adept inputs in conceptualizing the commentary, analysing the data, interpreting and drafting the commentary. KS has given her final approval of the version to be published and agrees to be held accountable for all aspects of the work related to the accuracy and integrity.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.


Osteoarthritis (OA) is still a highly prevalent and disabling disease for which we do not have a cure. In the last decade, however, the unravelling of molecular mechanisms controlling joint homeostasis, the advances in targeting technologies, and the improvement of animal models allowing the use of mouse genetics has led to the identification of molecular targets that, in animal models,et al9In this issue of the journal, Vasheghani The authors previously reported that cartilage-specific disruption of the gene encoding for the transcription factor PPARγ results in spontaneous OA in mice.The same group12Therefore they then tested the hypothesis that the worse OA outcome in PPARγ KO mice was due to enhanced mTOR signalling.mTOR is the catalytic subunit of two distinct molecular complexes, mTORC1 and mTORC2, which are differently regulated, have different downstream effectors  with the constrains of a pathologic environment , but that report on the activity of specific disease mechanisms to predict response to therapy.


Opioid-dependent participants were scanned three times, receiving reward from 50-mg intravenous heroin  during the first scan to generate expectation of the same reward at the second scan, during which they only received 0.1-mg intravenous heroin. There was no heroin injection during the third scan. Intravenous 50-mg heroin during the first scan induced pronounced effects leading to high levels of expectation at the second scan. There was no detectable increase in striatal dopamine levels to either heroin reward or expectation of reward. We believe this is the first human study to examine whether expectation of heroin reward increases striatal dopamine levels in opioid addiction. The absence of detectable increased dopamine levels to both the expectation and delivery of a heroin-related reward may have been due to the impact of substitute medication. It does however contrast with the changes seen in abstinent stimulant users, suggesting that striatal dopamine release alone may not play such a pivotal role in opioid-maintained individuals.The rewarding properties of some abused drugs are thought to reside in their ability to increase striatal dopamine levels. Similar increases have been shown in response to expectation of a positive drug effect. The actions of opioid drugs on striatal dopamine release are less well characterized. We examined whether heroin and the expectation of heroin reward increases striatal dopamine levels in human opioid addiction. Ten opioid-dependent participants maintained on either methadone or buprenorphine underwent [ The euphoric effect of stimulants has been shown to correlate with striatal dopamine release, and increases in dopamine have also been reported with alcohol, nicotine and cannabis . Consist11C]raclopride positron emission tomography (PET) imaging  interval between first and second PET scans 14.0 ± 7.6 days; mean ± SD interval between second and third PET scans 12.5 ± 7.4 days]. Each PET scan occurred at a similar time of day and participants were over 24 hours after their last dose of methadone or buprenorphine, which they deferred taking until after the scan. At the first (‘reward’) PET scan, participants were told they would receive an injection of pharmaceutical heroin , although the dose was undisclosed as in our previous study  on a separate day, with a Philips 1.5T Gyroscan Intera scanner  to produce structural images for reference.Each participant underwent three [11C]raclopride administered as an initial intravenous bolus (922 ml/hour for 60 seconds) followed by a constant infusion (8.8 ml/hour) for the remainder of the scan , with an axial field of view of 15.5 cm. A 10-minute transmission scan was acquired prior to each emission scan to correct for tissue attenuation. Dynamic emission scans were acquired in three-dimensional mode using a standard acquisition protocol  running in Matlab® . Accounting for radioactive decay, we previously reported [11C]raclopride equilibrium stability of 0.06% per minute over the equilibrium period using this bolus infusion protocol (For the region of interest (ROI) analysis, the inverse deformation parameters obtained from the segmentation process were applied to a functional striatal atlas (ND) values for [11C]raclopride in each striatal region were calculated as the ratio of striatal counts to cerebellar counts, minus 1, over the steady-state time periods  to create parametric BPt-tests were two-tailed. Paired samples t-tests were used to compare subjective measures. Correlations between parametric data were assessed using Pearson's product–moment correlation coefficient. Differences in early and late steady-state phases within and between different scans were investigated using repeated measures analysis of variance (ANOVA), with striatal region as a within-subject factor. Heterogeneity of covariance was tested with the Mauchly sphericity test and degrees of freedom modified using the Greenhouse–Geisser adjustment, where appropriate.All statistical comparisons, apart from voxelwise analyses, were performed using SPSS 18.0 . All t-test. A height threshold of P < 0.001 and volume-corrected cluster threshold of P < 0.05 were used for statistical significance  and one buprenorphine maintenance (5 mg/day). The mean ± SD length of prior opioid use was 13.9 ± 6.3 years. All participants were tobacco smokers and had last smoked cannabis more than 2 months prior to imaging. Six participants had no recent alcohol consumption, with the remaining participants consuming 8–18 units (64–144 g) of alcohol per week. Occasional stimulant use was reported by all participants, with last use over 2 years previously for all but two . Mean ± SD BDI and SSAI scores did not differ significantly between scans and were as follows: reward scan , expectation scan  and no-drug scan . The mean ± SD STAI score was 43.7 ± 11.3. These scores reflect only mild symptoms of anxiety and depression.Eleven opioid-dependent individuals started the three-scan protocol; however, one withdrew after the first scan due to subsequent failure of raclopride BPND values in any striatal ROI. Two comparisons were undertaken: [11C]raclopride BPND in early and late steady-state phases within the reward scan , and between early and late steady-state phases in the reward and expectation scans . In addition there were no significant correlations between VAS high scores and [11C]raclopride BPND values in any striatal region. Table ND values in each of the scans.No significant effect of expectation on 1CracloprND increased significantly from early to late steady-state phases  of intravenous heroin.11C]raclopride PET scan resulted in increased levels of expectation at the second scan in all participants. We enhanced the cues for the opioid-dependent participants compared with our previous study  of heroin did not result in significant increases in striatal dopamine in opioid-maintained individuals  to detect a 3.6% change in overall [11C]raclopride binding consistent with other studies that reported significant changes of 4–5% in [11C]raclopride binding with cue exposure , and secondly the recruitment of opioid-dependent individuals without co-morbid stimulant or benzodiazepine abuse. Consequently, to ensure a sufficient number of such participants were recruited to robustly test our hypotheses, the study took 5 years to complete. A further limitation of all [11C]raclopride PET studies is the uncertainty about what any differences in BPND represent. While changes are measurable following both pharmacological and psychological interventions, these may reflect either changes in synaptic dopamine concentration or in dopamine D2/3 receptor availability (While PET has been a highly useful tool for studying neurotransmitter systems in man, its sensitivity is limited. It is therefore possible that any dopamine release induced either by the expectation of heroin or heroin itself is too small or localized to be detected by current [In conclusion, we have demonstrated that striatal dopamine release does not appear to play a key role in mediating either the expectation or ‘high’ associated with heroin use in opioid-dependent individuals maintained on substitute medication. Given how many opioid-dependent individuals are in maintenance programmes, it is vital that we understand the impact of substitute medications on the dopaminergic system. In addition, there is growing evidence that dopamine is likely to play differing roles in opioid addiction compared with alcohol and cocaine addiction (