Binding of organic guests containing acidic or basic groups inside a water-soluble coordination cage host shows strong pH dependence. 4 M–1 in the neutral form (pH greater than 11), but the stability of the complex is reduced by three orders of magnitude when the guest is protonated at lower pH. Monitoring the uptake of the guests into the cage cavity was facilitated by the large upfield shift for the 1H NMR signals of bound guests due to the paramagnetism of the host. Although the association constants are generally lower, guests of biological significance such as aspirin and nicotine show similar behaviour, with a substantial difference between neutral (strongly binding) and charged (weakly binding) forms, irrespective of the sign of the charged species. pH-dependent binding was observed for a range of guests with different functional groups , so that the pH-swing can be tuned anywhere in the range of 3.5–11. The structure of the adamantane-1-carboxylic acid complex was determined by X-ray crystallography: the oxygen atoms of the guest form CH···O hydrogen bonds with one of two equivalent pockets on the internal surface of the host. Reversible uptake and release of guests as a function of pH offers interesting possibilities in any application where controlled release of a molecule following an external stimulus is required.A range of organic molecules with acidic or basic groups exhibit strong pH-dependent binding inside the cavity of a polyhedral coordination cage. Guest binding in aqueous solution is dominated by a hydrophobic contribution which is compensated by stronger solvation when the guests become cationic (by protonation) or anionic (by deprotonation). The Parkinson's drug 1-amino-adamantane (‘amantadine’) binds with an association constant of 10 These containers may be either organic capsules, often held together by hydrogen-bonding;2 or polyhedral coordination cages based on metal–ligand interactions.3A key goal of synthetic supramolecular chemistry is to control the use of weak, non-covalent interactions as the basis for planned self-assembly of combinations of molecular components with both  These include catalysis; sensing; drug delivery; and stabilisation of reactive intermediates. The consequences of guest binding can include alteration of the conformation of flexible guests due to space restrictions;4 stabilisation of otherwise unstable molecules;5 and – at its most sophisticated – size- and shape-selective catalysis of reactions in the cavity.6Within these classes of host there are now many well-characterised examples of guest binding with potential applications emerging in many areas where size/shape selective binding or transport of one specific guest can occur.control guest uptake and release is limited. Interactions between host and guest cannot usually be altered which means that the affinity of the guest for the host is fixed, and movement into/out of the cavity is based on a simple equilibrium over which we can exert little control. A particular guest may be displaced from a cavity by adding a competing guest that binds strongly; or concentrations of host and guest can be altered to adjust the position of an equilibrium without altering the equilibrium constant. In neither case is the strength of the interaction between host and guest modified.Despite the numerous examples of host–guest chemistry of container molecules, our ability to et al. have reported a triangular cage complex which binds planar guest molecules and then moves through cell membranes whilst carrying the payload.8 Crowley et al. recently described a cage which binds two molecules of the drug cis-platin [cis-PtCl2(NH3)2] via H-bonding interactions: however removal of the guest  requires decomposition of the host cage.9 Clever et al. have prepared a cage in which (reversible) photoinduced rearrangement of the structure, which incorporates photochromic units in the ligands, resulted in the guest being ejected.10 Fujita et al. reported how a redox change of a ferrocene guest in a cage cavity – switching the guest between neutral and cationic forms – resulted in reversible uptake and release of the guests.11 Both Nitschke12 and Fujita13 have shown how simple basic guests  are ejected from the cavity of a host cage host following protonation, allowing the use of a pH swing to control uptake/release in isolated cases. An interesting variant on this is the complete disassembly/re-assembly of a cage at different pH values which is associated with release/re-uptake of the guest.14 These existing examples can be conceptually separated into those that require rearrangement or decomposition of the host cage to liberate the guest, and those in which it is changing the charge on the guest that is the basis of uptake and release.It would be highly desirable therefore to find some external stimulus that can reversibly increase or decrease the affinity of a guest for its host, so that guest uptake and release can be triggered on demand. A few examples of such uptake/release do exist. Therrien This handful of disparate examples shows how the development of a mechanism for controlled uptake and release of guest molecules from containers according to an external stimulus is an important goal. A fully reversible uptake/release switching mechanism that can be applied to a wide range of guests under a wide range of conditions will make a major contribution to the development of useful functions from supramolecular assemblies in fields from medicine to catalysis.8L12]16+ coordination cages. These cages contain a Co(ii) ion at each vertex and a ditopic bridging ligand L (containing two chelating pyrazolyl-pyridine termini) spanning each of the 12 edges 12]16+ cage  showed a strong pH dependence. The use of pH changes to control supramolecular assemblies in many ways is well established.19 Prominent examples include changing the conformation of rotaxanes by protonation/deprotonation of specific sites on the axle;20 pH-induced disassembly of amphiphilic containers as a mechanism for drug release;21 and reversible control of the assembly/disassembly of crown ether/ammonium H-bonded systems using a pH swing.22The importance of the hydrophobic effectKa values, from the cavity of a coordination cage. Some of the guests have biological significance including use as prescription drugs.Accordingly we report here the results of a study showing how a pH swing can be used as the basis of reversible uptake/release of a range of guests, spanning a wide range of p8(Lw)12]16+ (hereafter denoted H) in water showed that simple substituted adamantanes such as adamantanone and 1-acetyl-adamantane bound well (K > 104 M–1), as the adamantyl group is a good size/shape match for the pseudo-spherical cavity of H in addition to having a high hydrophobic surface area.16 As we reported before, the paramagnetism of [Co8(Lw)12]16+, arising from the presence of high-spin Co(ii) ions, acts as a shift reagent which disperses the 1H NMR signals over a range of ca. 200 ppm. This makes it easy to separate the signals for free cage and the cage/guest complex under slow-exchange conditions, and integration of these signals at known concentrations of host and guest provides the association constants.Our recent work on binding of hydrophobic guests in the cavity of , the signals due to protonated free guest move to a lower chemical shift as the pH increases, and the protonated 1-aminoadamantane cation becomes deprotonated. Finally, in the region between –8 and –10 ppm [column (c)], the signals due to bound guest have a negative chemical shift because of the proximity to the eight paramagnetic metal ions surrounding the cavity. At pH 7 there is no measurable bound guest; as the pH is raised, the amount of bound guest steadily increases as the free 1-amino-adamantane cation is deprotonated, and the neutral form enters the host cavity.We extended the search to other substituted adamantanes, and were initially surprised to find no evidence for binding of 1-aminoadamantane. On addition of an excess of guest to a sample of viz. the proportion of cage occupied as a function of pH (in red), and the chemical shift of the most intense 1-aminoadamantane signal as a function of pH .i.e. in the presence of excess guest – occupancy of the cavity goes from negligible to complete (within the limits of error of the NMR measurements), and the pH at which this process is 50% complete is the same as the pKa of 1-aminoadamantane. In other words, the uptake and release of the guest from the cage cavity is driven by the deprotonation/protonation of the guest. Integration of the signals due to free and bound host at different concentrations of guest, during separate NMR titrations of H with 1-aminoadamantane at fixed pH values of 7 (weak binding limit) and 12 (strong binding limit), gave a binding constant of 1.0(3) × 104 M–1 for neutral 1-aminoadamantane and 13(7) M–1 for the protonated form, which is a change of three orders of magnitude and corresponds to a difference of 17 kJ mol–1 between the binding free energies of the neutral and cationic forms. The process is fully reversible.It is clear that under these conditions – Ka of 5.1, so the pH at which the host–guest interaction is switched on/off should be different. At pH values <5 there is a slight drift in the 1H NMR signals from the cage as a function of pH, due to deprotonation of some of the 24 externally-directed hydroxyl groups which are relatively acidic due to the high positive charge on the cage. However, binding of 1-adamantane-carboxylic acid is again in slow exchange on the 1H NMR timescale, so discrete signals were observed for free and bound host, and these were integrated to obtain the association constants for the neutral and deprotonated forms of the guest  × 104 M–1], than in its anionic state [K = 9.0(5) × 102 M–1]. This is the converse of what might be expected on purely electrostatic grounds given the positive charge of the cage, which means that any electrostatic interaction between host and guest is less significant than solvation effects. The carboxylate anion is bound more weakly simply because it is more hydrophilic and better solvated in water than the neutral carboxylic acid. To test this hypothesis, we measured the association constants for 1,3-adamantane-dicarboxylic acid over the same pH range. For the neutral diacid form at pH 3, K = 2.3(4) × 105 M–1, whereas for the dianionic form at pH 8, the association constant was too low to measure at the accessible concentrations (K < 300 M–1).Ka values of adamantane-1,3-dicarboxylic acid were measured by 1H NMR pH titrations as 4.8 and 5.9, so we can be confident that the binding constants measured at pH 3 and 8 correspond to the neutral and dianionic forms of the guest, respectively. High concentrations of the dianion at pH 8 resulted in decomposition of the cage, limiting our estimate of the binding constant of the dianionic form to <300 M–1.‡The two p For 1-adamantane-carboxylic acid, deprotonation results in a loss of binding free energy (ΔΔG) of 11 kJ mol–1, which must principally be associated with improved solvation of the free guest in water in its charged state, and the effect is larger (at least 16 kJ mol–1) for the diacid.1-Adamantane-carboxylic acid binds two orders of magnitude more strongly in its neutral state . The pKa of isoquinoline is 5.5, and it was possible to measure the association constant of the protonated form at lower values of pH: K = 10(2) M–1, which is a change of three orders of magnitude in the association constant (ΔΔG = 18 kJ mol–1). The behaviour of other molecules, some of which are of biological interest, as switchable guests is summarised in Ka = 8.1) binds with K = 81(20) M–1 in the neutral form, but when the tertiary amine group was protonated no evidence of binding was observed. The sedative and anaesthetic molecule detomidine24 has an imidazole moiety as the ionisable group (pKa = 7.1), and the association constant drops from K = 70(30) M–1 for the neutral form to undetectably small following protonation. Binding of neutral aspirin (pKa = 3.5) occurs with K = 120(30) M–1, and this also falls to undetectably small for the deprotonated anionic form. None of these examples matches the high binding affinity of the adamantane-based guests, presumably due to a less optimal shape/size match for the host cavity and less hydrophobic character, but they still show pH-induced switching of binding.Starting from the family of bicyclic guests that we know can be accommodated in the cavity of the cage,G – i.e. the difference in free energy of binding between neutral and charged forms – is significantly larger  than with 1-adamantane-carboxylic acid (ΔΔG = 11 kJ mol–1). This presumably reflects an additional electrostatic contribution to complex stability which depends on the charge of the guest. Thus we expect an attraction between H and adamantane-1-carboxylate which slightly stabilises the complex with the anionic guest and gives a smaller value of ΔΔG, but a repulsion between H and protonated isoquinoline/protonated 1-aminoadamantane which slightly destabilises the complexes and gives a larger value of ΔΔG. The consequence is a two order-of-magnitude swing for the pH-dependent binding constant of 1-adamantane-carboxylic acid but a three order-of-magnitude swing for 1-aminoadamantane and isoquinoline. Improved solvation of the charged form of the guest irrespective of sign is the dominant factor in determining ΔΔG, but an additional electrostatic contribution is also evident.We note that for the two basic guests 1-aminoadamantane and isoquinoline for which association constants could be measured in both neutral and protonated forms, the value of ΔΔH with adamantane-1-carboxylic acid,8(Lw)12](BF4)16·(C11H16O2): C371H328B16Co8F64N72O26, M = 8071.43 g mol–1, monoclinic, space group C2/c, a = 27.3936(7), b = 39.1227(10), c = 41.964(3) Å, β = 107.152(8)°, U = 42 973(4) Å3, Z = 4, ρcalc = 1.248 g cm–3, T = 100(2) K, λ (Mo-Kα) = 0.71075 Å, μ = 0.393 mm–1. 147 295 reflections with 2θmax = 55° were merged to give 49 124 independent reflections (Rint = 0.052). Final R1 [for data with I > 2σ(I)] = 0.163; wR2  = 0.469. The data collection was performed by the EPSRC National Crystallography Service at the University of Southampton  and long (single) distinction between the C–O bonds, the presence of disorder of the entire guest over two orientations – plus the additional possibility of C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="34" y-size="34" xml:space="preserve"> 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000	</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="16.000000pt" height="16.000000pt" viewBox="0 0 16.000000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 1440 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z M0 960 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z"/></g></svg>O/C–OH disorder within each orientation – means that we cannot draw any conclusion from the bond lengths.¶The distinction is not crystallographically obvious as extensive disorder of the tetrafluoroborate anions in the structure means that not all of them could be located – so we cannot use charge balance considerations to determine whether or not the guest is protonated. Although the two C–O bond distances of the carboxylic acid (or carboxylate) group appear to be approximately equivalent,  As is normal for cage complexes of this type, weak scattering resulted in a relatively high R1 value of 16%, which means that detailed analysis of structural minutiae is not appropriate, but the formation of the complex and its key structural features are clear  between the carboxylic acid oxygen atoms and some of the naphthyl and methylene protons on the interior surface of the host  host and guest mean that the ΔΔG values, and hence the efficacy of the pH swing at modulating guest uptake and release, are slightly larger between neutral/cationic guest pairs than between neutral/anionic guest pairs. The pH swing works over a range of values, from 3.5–11 depending on the pKa of the guest, with several different functional groups , and the cage is remarkably stable over this entire pH range. Some of the guests investigated  have been used as drugs for which the ability to control uptake and release by an external perturbation is clearly a desirable target.We have demonstrated reversible pH-dependent uptake and release of several types of guest molecule into/out of the hydrophobic central cavity of a water-soluble coordination cage host; a graphical summary of the results is shown in


It is not clear which is the best anthropometric indicator to predict metabolic syndrome (MetS) in adolescents. Our objective was to identify the predictive power, with respective cut-off points, of anthropometric indicators associated with the quantity and distribution of body fat for the presence of MetS and to determine the strength of the association between the proposed cut-off points and MetS in adolescents.International Diabetes Federation. Predictive performance was described through analysis of Receiver Operating Characteristic (ROC) curves with a 95% confidence interval. The most accurate cut-off points were identified through sensitivity, specificity and Area Under the Curve (AUC) values.The sample consisted of 1035 adolescents (565 girls and 470 boys) aged between 12 and 20 years. Four anthropometric indicators were considered: waist circumference (WC), body mass index (BMI), waist-height ratio (WHtR), and conicity index (C-Index). MetS was defined according to the criteria of the The four anthropometric indicators presented significant AUCs close to 0.70. At younger ages (12-15 years) the girls presented a statistically greater capacity to discriminate MetS; however, at more advanced ages (16-20 years) both sexes presented similar AUCs. Among the anthropometric indicators investigated, regardless of sex and age, the WHtR showed the highest discriminant value for MetS, while the C-Index demonstrated a significantly lower capacity to predict MetS. The AUCs equivalent to WC and BMI did not differ statistically. The proposed cut-off points for WHtR  presented the highest values of sensitivity and specificity, between 60% and 70%, respectively.Considering that the best AUC was found for WHtR, we suggest the use of this anthropometric indicator, with the cut-off points presented herein, for the prediction of MetS in adolescents with characteristics similar to the study sample. Metabolic syndrome (MetS) refers to a set of risk factors that, when altered, may increase the chances of developing cardiovascular diseases and diabetes mellitus –3. The rThe literature presents strong evidence that cardiometabolic alterations, manifested in adulthood, result from complex interactions between a variety of risk factors that may originate in childhood and adolescence , 5. TherHowever, as the diagnosis of MetS involves invasive laboratory tests to determine the plasma lipid profile and glycemic rate, its inclusion on a large scale in the routine monitoring of the health status of adolescents is complex. In this sense, efforts have been directed in the attempt to indicate more affordable and inexpensive alternatives for epidemiological tracking and, thus, direct specific procedures to those at risk of developing MetS –10.Results found in previous studies suggest that excess body fat is characterized as an important contributor to triggering MetS in the pediatric population –13. In tConsidering that the anthropometric indicators associated with the quantity and distribution of body fat are easy to handle, are not invasive and have low cost, it is important to identify the predictive power of each anthropometric indicator and its respective cut-off points for detection of MetS in young population. However, it should be pointed out that this referral does not seek to replace medical intervention, since it does not exclude the need to identify the individual components to confirm the diagnosis of MetS. Screening, when performed in environments with high concentrations of young people, such as schools, can reach a high number of adolescents, especially those who have difficulty accessing or do not attend the health system. Thus, once the adolescents most likely to present MetS have been identified, they can be referred for specialized medical follow-up.Some studies have evaluated the predictive capacity of anthropometric indicators to detect MetS; however, conflicting data were found. Kelishadi et al.  found WCIn addition to the divergences found among the results of the studies, it is noteworthy that the C-Index has not been tested for its predictive ability to detect MetS in adolescence. In adults the C-Index presented superior performance to other anthropometric indicators to predict cardiovascular risk .Therefore, the objectives of the present study were to identify the predictive power, with respective cut-off points, of four anthropometric indicators associated with the quantity and distribution of body fat  for the presence of MetS and to determine the strength of the association between the proposed cut-off points and MetS in adolescents.The study is linked to a larger project, which aimed to identify the prevalence of MetS and associated factors in adolescents. For this, a descriptive cross-sectional survey was carried out with a population base involving schoolchildren from the city of Jacarezinho, Paraná, Brazil. Data collection extended from August to November 2014. The intervention protocols used followed the Declaration of Helsinki and were approved by the Research Ethics Committee of the Universidade Norte do Paraná – UNOPAR (Opinion 1.302.963).deff) as equivalent to 1.5, estimating, therefore, a minimum sample of 1000 adolescents in school. However, the final sample used in the treatment of information was composed of 1035 adolescents (565 girls and 470 boys). In the present study, the statistical power of the sample, stratified by sex (565 girls and 470 boys), was calculated a posteriori and enabled identification with 80% power, a significance of 5%, and areas under the ROC (Receiver Operating Characteristic) curve of at least 0.53 and 0.56 for girls and boys, respectively.The reference population included adolescents of both sexes, between 12 and 20 years of age, enrolled in public and private elementary schools (6th to 9th grade) and high school (1st to 3rd grade). Initially, the sample size was established to meet the primary objective of the project to identify the prevalence of MetS and associated factors, assuming a 95% confidence interval, a sampling error of 3 percentage points, and an increase of 10% to allow for eventual lost cases during data collection. In addition, considering that the sample planning involved conglomerates, we defined the effect of the sample design . The criteria adopted to exclude some adolescents drawn for the study were: (a) refusal to participate in the study; (b) not signing the Free and Informed Consent Form; (c) any health problem that temporarily or permanently prevented participation in the study; (d) using any type of medication that could induce changes in the study variables; (e) undergoing any type of specific diet; (f) pregnancy; and (g) non-attendance at school on the day scheduled to begin data collection. In these cases, a new draw was carried out to restore any sample losses.In order to determine the body weight measurements, an anthropometric scale with a 10 g definition was used, brand SECA , model 813, checked every ten weighings, while to carry out the height measurements an aluminum stadiometer was used with a 1 mm scale, brand SECA , model 213. The WC measurements were performed using a flexible anthropometric inelastic fiberglass tape with a 1 mm scale, brand SECA , model 203.Measurements of body weight, height, and WC were performed according to the recommendations of the World Health Organization . Each prFor the height measurements, the adolescent, barefoot, was placed on the base of the stadiometer, upright, with the upper limbs hanging beside the body, feet together, trying to maintain the posterior surfaces of the heels, pelvic girdle, shoulder girdle, and occipital region in contact with the measurement scale. The distance between the plantar region and the vertex was determined with the aid of a cursor. The adolescent remained in inspiratory apnea and their head was oriented in the Frankfurt plane parallel to the ground.WC measurements were determined with the adolescent standing, with a relaxed abdomen and arms beside the body. The anthropometric tape was positioned in the horizontal plane, so as to encircle the natural waist line, at the coincident point of the mean distance between the last costal arch and the iliac crest, in a firm manner; however, without skin compression. The reading was obtained at the end of a normal expiration.2). The WHtR was obtained by dividing the waist circumference measure by the height in centimeters [The BMI was calculated through the ratio between the body weight measured in kilograms and the height expressed in meters squared  and blood glucose, resting blood pressure (systolic and diastolic), and abdominal fat accumulation (waist circumference), according to the criteria proposed by the International Diabetes Federation (IDF) . In thisPlasmatic lipid and blood glucose measurements were performed by collecting 10 ml venous blood samples at the elbow crease after a 10-12 h fasting period between 07:00 and 08:00 a.m. The serum was immediately separated by centrifugation, and the HDL-cholesterol dosages were determined by the precipitating reactive method, serum triglycerides by the enzymatic glycerol method, and glycemia by the calorimetric enzymatic methodology.The systolic and diastolic arterial blood pressure levels were measured by the auscultatory method using a mercury column sphygmomanometer. With the adolescent sitting, after a minimum of 5 min of rest, blood pressure was measured in the left arm. The systolic blood pressure value corresponded to Korotkoff phase I and diastolic blood pressure to phase V, or the disappearance of sounds. Two measures were taken, considering the mean value of both measures for calculation purposes.two-way analysis of variance with interaction, accompanied by the Scheffe multiple comparison test.Statistical analysis was performed using SPSS software, version 22. For the analysis of continuous variables, procedures of descriptive statistics were used (mean ± standard deviation). As the treated variables presented normal distribution of data, the comparisons between sex (girls and boys) and age (12 to 15 years and 16 to 20 years) for the anthropometric indicators were performed using The predictive capacity, sensitivity, and specificity of the four anthropometric indicators  to identify the presence of MetS, accompanied by the respective 95% confidence intervals, were defined using the ROC curve, to establish cut-off points in diagnostic or screening tests . The AreCut-off points for each anthropometric indicator capable of predicting MetS were determined by the best balance between sensitivity and specificity. Thus, the main objective of the analysis is to determine the value at which sensitivity and specificity indicate a threshold that maximizes the true-positive rate, maintaining the lowest possible rate of false-positive cases. Possible significant differences between the properties of sensitivity, specificity, and AUCs were identified using McNemar’s statistical test .After determination of the cut-off points for each of the predictive anthropometric indicators of MetS, they were dichotomized based on their respective reference values. The prevalence ratios accompanied by the respective 95% confidence intervals, stratified by sex and age, were calculated using Poisson regression.International Obesity Task Force [Statistical information equivalent to the anthropometric indicators that characterize the sample selected for the study are provided in Table sk Force , the excThe performance of the anthropometric indicators as predictors of MetS is presented in Table After determination of the cut-off points, the strength of the association between each of the anthropometric indicators and the presence of MetS was verified. The prevalence ratios and their respective confidence intervals are presented in Table The present study investigated the ability of anthropometric indicators associated with the quantity and distribution of body fat to discriminate the presence of MetS in adolescents. The ability of the four anthropometric indicators to predict MetS in adolescents aged 12 to 20 years of both sexes was confirmed. However, when comparing the AUCs found for each of the anthropometric indicators, significant differences were identified, indicating different accuracy. The anthropometric indicator that showed the highest predictive capacity for MetS was WHtR, followed, in this order, by BMI, WC, and C-Index.It is not uncommon to find higher scores equivalent to anthropometric indicators associated with quantity (BMI) and centripetal body fat distribution  in boys and older adolescents , 10, 24.Corroborating with findings made available through a systematic review that synthesized data from approximately 100 surveys conducted in different regions of the world , the preIn the present study, with identical cut-off points for both sexes, although different for adolescents aged 12-15 years (0.46) and 16-20 years (0.48), the WHtR was indicated as the anthropometric indicator that best discriminates the presence of MetS. Both cut-off points indicated sensitivity and specificity values between 60% and 70%, which moderately minimizes false-positive and false-negative cases. However, a very uncommon way of analyzing the diagnostic capacity of specific cut-off points is by calculating the positive (PLR) and negative (NLR) likelihood ratios. In the case of the youngest group (12-15 years), the PLR was equivalent to 1.95 in girls and 1.86 in boys, suggesting that those adolescents with WHtR ≥0.46 may present approximately twice the chance of a positive diagnosis being true; while the NLR corresponded to 0.52 and 0.48, respectively, which is also close to twice the chance of a negative diagnosis confirming the absence of MetS. Among the older adolescents (16-20 years), The PLR was equivalent to 2.33 and 1.86, while the NLR corresponded to 0.43 and 0.54 for girls and boys, respectively.The study by Benmohammed et al.  using thOn the other hand, coinciding with the findings of the present study, other investigations have found predictive capacity for MetS through anthropometric indicators associated with the quantity and distribution of body fat. Jung et al. , also usThe disagreements between the studies that have been carried out regarding the anthropometric indicator with better predictive performance for MetS may perhaps be justified by the ethnic origin of the adolescents selected in the different studies. In this regard, the ethnicity of adolescents is capable of influencing the definition of the cut-off points of the anthropometric indicators associated with overweight and body fat and the individual components that make up MetS, which could impact on their prevalence . Thus, iReferring specifically to the cut-off points best able to predict MetS through the WHtR, a systematic review aimed at analyzing the potential of anthropometric indicators to predict cardiovascular disease indicated WHtR ≥0.50 as the most appropriate for both sexes . This cuUsing the cut-off points defined in the present study the four anthropometric indicators investigated were significantly associated with the presence of MetS. However, we highlight the higher probability of identifying MetS in girls than in boys. Girls with WHtR ≥ 0.46 (12-15 years) and WHtR ≥ 0.48 (16-20 years) demonstrated probabilities around three to three and a half times higher for MetS, while boys with WHtR scores above cut-off points were approximately twice as likely to present MetS. Previous studies have also found greater accuracy to identify MetS by means of anthropometric indicators among girls ; howeverTo our knowledge this is the first study performed with Brazilian adolescents to verify the performance of different anthropometric indicators associated with the quantity and distribution of body fat as predictors of MetS. Identifying MetS in pediatric populations is not routine in the clinical setting, except in specific situations, such as the presence of obesity and diabetes. In addition, the decrease in the frequency of medical consultations during adolescence reduce the possibility of early detection of metabolic alterations, and the lack of diagnosis, control, and treatment of these alterations may constitute a factor impeding the prevention of future cardiometabolic outcomes. The findings of the present study will enable, by means of simple and accessible procedures, the screening of the possible presence of MetS and, when appropriate, referral to a specialized service to confirm the diagnosis.On the other hand, the present study presents some limitations that must be considered. School-based sampling may weaken the representativeness of the adolescent population. However, it should be emphasized that adolescents from all the city’s educational networks were distributed proportionally in the schools selected for the study. Even taking into account the low refusal rate for anthropometric measurements and laboratory tests (≈ 8%), the possibility of selection bias cannot be ruled out, since it was not possible to compare the treated indicators between the adolescents participating and not participating in the study. It is also important to note that, since there is no universal criterion for defining MetS, we chose to use the criterion proposed by the IDF and, in this sense, estimates of the prevalence of MetS may vary according to the criterion used.The four anthropometric indicators investigated demonstrated ability to predict MetS in adolescents aged 12 to 20 years of both sexes. However, considering that the best AUC was found for the WHtR, we suggest the use of this anthropometric indicator, with the cut-off points presented herein, for the prediction of MetS in adolescents with similar characteristics to the study sample. In this sense, it is assumed that approximately three out of four adolescents with MetS can be correctly diagnosed, constituting, therefore, a reasonable alternative to be used in initial screening to identify adolescents at higher cardiometabolic risk.


A dynamic phenotypic change contributes to the metastatic progression and drug resistance in malignant melanoma. Nevertheless, mechanisms for a phenotypic change have remained to be addressed. Here, we show that Met receptor expression changes in a cell-autonomous manner and can distinguish phenotypical differences in growth, as well as in metastatic and drug-resistant characteristics. In metastatic melanoma, the cells are composed of Met-low and Met-high populations. Met-low populations have stem-like gene expression profiles, are resistant to chemotherapeutic agents, and have shown abundant angiogenesis and rapid tumor growth in subcutaneous inoculation. Met-high populations have a differentiated phenotype, are relatively resistant to B-RAF inhibitor, and are highly metastatic to the lungs. Met plays a definitive role in lung metastasis because the lung metastasis of Met-high cells requires Met, and treatment of mice with the Met-containing exosomes from Met-high cells facilitates lung metastasis by Met-low cells. Clonal cell fate analysis showed the hierarchical phenotypical changes from Met-low to Met-high populations. Met-low cells either showed self-renewal or changed into Met-high cells, whereas Met-high cells remained Met-high. Clonal transition from Met-low to Met-high cells accompanied changes in the gene expression profile, in tumor growth, and in metastasis that were similar to those in Met-high cells. These findings indicate that malignant melanoma has the ability to undergo phenotypic change by a cell-intrinsic/autonomous mechanism that can be characterized by Met expression. The incidence of malignant melanoma is increasing faster than that of other solid tumors . The 5-yAutocrine and paracrine growth factors secreted within a tumor microenvironment participate in metastasis and drug resistance in malignant melanoma , 4. In aMet/hepatocyte growth factor (HGF) receptor mediates tumor cell proliferation, survival, invasion, and metastasis –11. PrevIn the present study, we found that there are different populations in metastatic melanoma cells, and these are characterized by different cell-surface Met expressions. Cells with Met-low and Met-high expressions have different profiles in gene expression, tumorigenicity, growth, and metastasis. Cell-surface Met expression was found to be in dynamic equilibrium and regulated by hierarchical and cell-autonomous changes between Met-low and Met-high populations.Met mRNA levels were much higher in the Met-high cells than those in the Met-low cells . Consistently, the dual analysis of the cell surface Met and Kit indicated the cells were composed of two major populations characterized by Kit-high/Met-low and Kit-low/Met-high  and dacarbazine , because xenobiotic transporters sistance , 27 werein vivo, Met-low and Met-high cells obtained after cell sorting were inoculated in the left and right subcutaneous regions, respectively, per each of the C57BL/6 syngenic mice. Both Met-low and Met-high cells formed tumors at a rate of 100%, when subcutaneously inoculated at 100 cells/site (n = 4). However, Met-low cells formed tumors more efficiently than Met-high cells when subcutaneously inoculated at 10 cells/site (100% (11/11) in Met-low cells and 72.7% (8/11) in Met-high cells). Tumors from Met-high cells were highly pigmented, while the tumors from Met-low cells were mostly non-pigmented with the exception of partially pigmented regions . These results suggest that the formation of functional blood vessels may be attributable to the higher growth potential of tumors from Met-low cells.The tumor volume in the Met-low cells became much larger than in the Met-high cells 28 days post-inoculation Figure , indicatTo know whether selective suppression of Met might influence tumor growth properties, Met-high cells were subjected to the stable expression of shRNA targeting Met (sh-Met) or non-targeting shRNA (Non-target). The knockdown of cell-surface Met expression was confirmed by flow cytometry . When Me5 cells per mouse were injected into the tail veins , and the incidences of metastasis to the lungs were analyzed 21 days later. The number of lung metastasis was much higher in Met-high cells (39.1 ± 31.4 per mouse) than in Met-low cells (8.3 ± 10.2 per mouse) . Histological analysis indicated that tumors from Met-low cells were not pigmented, whereas tumors from Met-high cells were highly pigmented , then 10 Figures   were subjected to analyses for a gene expression profile, tumor growth and metastasis. Microarray analysis indicated that the expression of the characteristic genes in the transitted cells showed a transitional profile or closer profile to that of Met-high cells than to Met-low cells Figure . In subcMelanomas are highly heterogeneous tumors, but the mechanisms for a phenotypic change remained mostly unknown . AnalysiMitf genes [Previous studies have indicated that Met functions in cancer stem cells in several cancer cell types, such as colon cancer, prostate cancer, and glioblastoma –31. HGF tf genes , 25.The metastatic characteristics of Met-low and Met-high melanomas differed in organotropism. Met-high cells metastasize mostly to the lungs, whereas Met-low cells metastasize to the liver, kidney, and other tissues. The low frequency of multi-organ metastasis of Met-low cells suggests that only a small population of Met-low cells can metastasize to multiple organs. Identification and characterization of theses cells could lend a better understanding of the multi-organ metastatic potential of melanomas. On the other hand, our results indicate that the highly metastatic potential of Met-high cells functionally required Met expression. Consistent with the previous report describing that exosome-packaged Met from B16-F10 melanoma forms a pre-metastatic niche in lungs [Tumor-associated angiogenesis differs between Met-low and Met-high melanomas. Tumors from Met-high cells developed vascular structures without pericytes. This might be explained by the low expression of platelet-derived growth factor (PDGF) and angiopoietin-2 (Angpt2), because the former facilitates the recruitment of pericytes and the latter induces sprouting angiogenesis , 35. On Malignant melanoma is one of the most chemoresistant tumors, and why melanomas are particularly insensitive to chemotherapeutic agents remains poorly understood , 37. XenFinally, cell-intrinsic and hierarchical change in Met expression and the associated gene expression profiles could be a mechanism that might help explain the intrinsic diversity and plasticity in tumor growth, metastasis, and drug resistance in malignant melanomas. Elucidation of the mechanism by which Met expression in individual cells is regulated overall in a certain equilibrium between Met-low and Met-high cell populations may facilitate further understanding of the intrinsic nature of malignant melanomas.Cells were obtained from the American Type Culture Collection. B16-F10 cells were cultured in RPMI1640 medium supplemented with 10% (v/v) fetal bovine serum. A2058 cells were cultured in DME medium supplemented with 10% (v/v) fetal bovine serum and 2 mM glutamine. For analysis of Met expression in clonal cell populations, cells sorted by flow cytometry were first cultured on a 96-well plate at a density of one cell/well, and were then cultured for 3–4 weeks. Mouse Met shRNA sense sequences were obtained from the Broad Institute TRC shRNA library , or an anti-mouse Kit antibody . Secondary antibodies conjugated with phycoerythrin (PE) or allophycocyanin (APC) were used (Santa Cruz). Isotype controls were used to set the background. Live-cell selection was accomplished using 7-AAD (BD biosciences). Cells were analyzed and/or sorted on a FACS canto II, a FACS Aria II (Becton Dickinson) or with a JSAN cell sorter . Cell cycles were determined using a Cell-Cycle Phase Determination Kit . Apoptotic cells were stained using an Annexin V-FITC Apoptosis Kit .Microarray analyses were performed using the Whole Mouse Genome  Oligo Microarray, according to the Agilent 60-mer Oligo Microarray Processing Protocol (Agilent Technologies). Total RNA samples (200 ng) were used to prepare Cy3-labeled cRNA using a Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies). Fluorescence-labeled cRNAs were purified using an RNeasy RNA Purification Kit . Two independent RNA samples were used to confirm the reproducibility of the microarray analyses. The images were analyzed using Feature Extraction Software (Ver. 10.7.3.1) and GeneSpring GX 11.5 software (Agilent Technologies). Normalization was performed as follows: (i) intensity-dependent Lowess normalization; (ii) data transformation, with measurements set to ≤0.01; (iii) per-chip 75th-percentile normalization of each array; and, (iv) per-gene: normalized to the median of each gene. Genes differently expressed more than twice between Met-low and Met-high populations were selected and used for the gene ontology enrichment analysis. Gene ontology (GO) enrichment analyses were performed using DAVID Bioinformatics Resources 6.7. The raw and processed data were deposited in the Gene Expression Omnibus (GEO) database (access ID: GSE69741).5 cells in 100 μl of PBS were injected into the tail veins, and the degrees of metastases were analyzed 21 days later. All animal experiments were performed in accordance with the animal experiments guidelines of Kanazawa University.Five week-old female C57BL/6 mice  were used. Met-low and Met-high cells were prepared via 2 successive cell sortings. For subcutaneous growth, 10, 50, or 100 cells in 50 μl culture medium were mixed with 50 μl of growth factor-reduced matrigel, and subcutaneously injected bilaterally. Tumor volume was analyzed on day 35 or within 35 days after the inoculation unless otherwise specified. For metastasis assay, 10® M.O.M.™ Immunodetection Kit . Sections were analyzed using a Biozero BZ-9000 .For immunostaining of Met, frozen sections were fixed with methanol, tissue sections were incubated with PBS containing 3% bovine serum albumin for 30 min, washed with PBS, and incubated overnight with rabbit anti-Met antibody  in PBS containing 0.5% bovine serum albumin and 0.1% Tween-20 at 4 °C. After washing, sections were incubated with EnVision+ System-HRP Labeled Polymer . Immunocomplexes were visualized using an IMMPACT DAB kit  and nuclei were stained with hematoxylin. For blood vessel staining, frozen sections were fixed with methanol, then treated with rat anti-CD31  and mouse anti-α-smooth muscle actin  antibodies were diluted via a VectorTotal RNA was prepared using Sepasol RNA , and RNA were reverse transcribed using Superscript III Reverse Transcriptase and Oligo(dT) primer (Invitrogen). Quantitative PCR reactions were performed in triplicate using SYBR-green dye (Roche Diagnostics) with the 7500 Sequence Detection System . The transcription level of β-actin was used for normalization. The primer sequences are listed in Table ®  was used for the dilution of antibodies.Western blotting was performed as described elsewhere . Brieflyt-test or a Mann-Whitney's U test using Prism 6 software. A P < 0.05 was considered significant.Error bars represent the means ± standard deviation. Mouse experiments were performed in duplicate using at least 4 mice per treatment group. Multiple-group comparisons were analyzed using either a Tukey's test or ANOVA. Two group comparisons were analyzed using either a Student's


Given the difficulty in obtaining adequate tissue in NSCLC, we investigated the utility of circulating tumor cells (CTCs) for MET status assessment in NSCLC patients. We used two platforms for CTC capture, and assessed MET expression in CTCs and matched-bronchial biopsies in patients with advanced-stage III/IV lung adenocarcinoma. Baseline peripheral blood was collected from 256 advanced-stage III/IV NSCLC patients from Genentech clinical trials, and from 106 patients with advanced-stage III/IV lung adenocarcinoma treated at the Department of Pneumology, Pasteur Hospital, Nice. CTCs were enriched using CellSearch (Genentech), or ISET technologies . MET expression was evaluated by immunofluorescence on CellSearch, and by immunocytochemistry on ISET-enriched CTCs and on matched FFPE tissue sections . CTCs were detected in 83 of 256 (32%) patients evaluated on CellSearch, with 30 samples (12%) exhibiting ≥ 5 CTCs/7.5 ml blood. On ISET, CTC were observed in 80 of 106 patients (75%), and 79 patients (75%) exhibited ≥ 5 CTCs/4 ml blood. MET expression on ISET CTCs was positive in 72% of cases, and the MET expression on matched-patient tissue was positive in 65% patients using the Onartuzumab IHC scoring algorithm (93% concordance). Quantification of MET expression using H-scores showed strong correlation between MET expression in tissue and CTCs . MET status in CTCs isolated on ISET filters from blood samples of advanced-stage NSCLC patients correlated strongly with MET status in tumor tissue, illustrating the potential for using CTCs as a non-invasive, real-time biopsy to determine MET status of patients entering clinical trials. EGFR gene mutations (TKI), EML4–ALK fusion and ROS1 translocations, have proven marked treatment responses being more effective than conventional chemotherapies in advanced NSCLC patients [Non-small cell lung cancer (NSCLC) accounts for approximately 80% of lung cancers and remains a major cause of cancer death worldwide . In the patients , 4. Howepatients . Dysregupatients –8. The Mpatients . Overexppatients –14. Sevepatients .ALK-rearranged NSCLC patients, it has shown activity in a subgroup of NSCLC patients that lack an ALK translocation but overexpress MET, or carrying de novo MET gene amplification [MET receptor tyrosine kinase has emerged as a potential therapeutically relevant target in NSCLC , 17. A nfication –22.Eligibility of patients to targeted therapies relies on diagnostic assays performed on a tumor biopsy. This invasive procedure is associated with a relative high risk of morbidity, and inoperable patients can be deprived from potentially more efficient therapies . MoreoveCirculating tumor cells (CTCs) represent an accessible, non-invasive surrogate tissue that allows access to biomarker assessment in vulnerable lung cancer patients for whom tissue biopsies are inaccessible or extremely difficult to perform and to repeat . Among tTo investigate the utility of a liquid biopsy to assess a patient's lung tumor's MET status, here we evaluated the prevalence of MET expression in CTCs using 2 different CTC platforms, CellSearch and ISET, and compared MET expression in CTCs and matched tumor tissue in a retrospective cohort of 80 advanced-stage NSCLC patients.Baseline blood samples from 256 Stage III/IV NSCLC patients were evaluated for CTC enumeration on the CellSearch platform. CTC enumeration ranged from 0 to > 200 CTCs in 7.5 ml blood. One or more CTCs were observed in 83 patients (32%), with 30 samples 12%) exhibiting ≥ 5 CTCs/7.5 ml blood Figure , Table 12% exhibi°C for up to 180 days, or immediately stained with MGG on 4 out of the 10 filter-spots (see Methods). These 4 spots, enriched in the filtrate from 4 ml blood, were examined for the presence of CNHC. CTC enumeration was computed as the number of CNHC-MF and CNHC-UMF cells identified in 4 ml blood. CNHC-MF and/or CNHC-UMF were detected in 80 of 106 (75%) patient samples examined. 79 of the 80 CTC-positive samples exhibited more than 5 CTCs per 4 ml blood  were chemotherapy naïve, and 7 (7%) had neoadjuvant chemotherapy. Blood samples were processed within one hour of collection using the ISET filtration system. Filters were either stored at −20d Figure , with a A representative example of a circulating cell with malignant features, characterized by a large size (diameter larger than 20 μm), large nuclei and presence of nucleoli is shown in Figure The ICC assay to evaluate MET expression on CTCs was developed using a panel of human cell lines that express low, medium and high levels of MET. Cell-lines spiked into healthy donors’ blood were enriched on ISET filers and stained for MET. Staining conditions were optimized to result in concordant MET scores for cell-blocks (IHC) and the corresponding blood spike-ins (ICC). The progressive increase in MET staining intensities from low- to high-MET expressing cells observed in cell blocks was paralleled by a progressive increase in CTC staining intensity in the corresponding spike-in samples . Next, wMET IHC was evaluated in FFPE tissue samples from patient-matched biopsies. Applying the Onartuzumab algorithm, MET patient status was positive in 49 of 75 (65%) patients’ tissue samples Figure . CompariTo get a more finely graded picture of MET expression in the tumor tissue and CTCs, we also computed MET expression using an H-scoring system that takes into consideration the staining intensity and the proportion of CTCs exhibiting each MET score. Comparison of patient-matched tumor-tissue and blood showed a linear correlation between MET H-scores in tumor-biopsies and CTCs Figure , with a P-value = 0.159; gender, P-value = 0.088; smoking status, P-value = 0.280; tumor size, P-value = 0.089). At a median follow up of 29 months, 46 patients had progressed and 26 patients had died. MET expression in tumor tissue or CTCs did not correlate with PFS in univariate survival analysis  and NCT01496742 studies , and the even smaller number of patients with MET expression in CTCs precludes any robust analysis of the value of CTCs in these studies.In NSCLC patients evaluated for CTCs on CellSearch, a prognostic value of CTC counts was reported using a cutoff of 5 CTCs . The smavs. ISET) and MET evaluation methodology (immunofluorescence vs. immunohistochemistry), and also in blood stabilizing agents and time from blood collection to processing. In the CellSearch-based approach used for Genentech trials’ samples, blood was collected in CellSave tubes and shipped to a central lab for CTC enumeration, a process that lasted from 18 to more than 96 hours. In the ISET-based approach used for the Pasteur Hospital samples, blood was collected in EDTA tubes and processed for CTC enumeration within 1 hour of collection. We found that the ISET approach identified a higher proportion of CTC-positive patients than the CellSearch approach. This was consistent with previous reports demonstrating high sensitivity for CTC enrichment using the ISET methodology in blood samples from NSCLC patients [vs. CellSearch systems could also reflect the distinct CTC enrichment methodologies of the two platforms: antigen agnostic on the ISET platform, and EpCAM-dependent CTC enrichment on CellSearch. Non-epithelial or mesenchymal CTCs in blood from NSCLC patients would be detected uniquely on the ISET platform, but escape the antigen-dependent enrichment method of the CellSearch system [In this study we evaluated the potential utility of CTCs for assessment of MET in advanced NSCLC patients. We tested two approaches for CTC analysis that differed in the platform for CTC enrichment  [The relationship between MET overexpression and the outcome of NSCLC patients remains controversial, and may depend on the primary antibody used for MET assessment, cutoff criteria, and selection of samples (e.g. biopsy samples) , 29. Thesamples) . These fIn conclusion, we showed that CTCs from NSCLC patients are successfully detected on the ISET platform, and that MET status in ISET-captured CTCs correlate strongly with MET status in tumor tissue. This study provides a proof-of-concept for the potential use of CTCs as a sensitive and specific diagnostic testing of MET protein expression in patients with advanced-stage NSCLC that might be eligible for targeted therapies and could be of use to monitor therapy resistance. We are now expanding the CTC analysis to other biomarkers with relevance for lung therapeutics.For the analysis of CTCs on the CellSearch system, peripheral blood samples were obtained from 256 Stage IIIB/IV NSCLC patients enrolled in Phase II clinical trials NCT01519804 (squamous NSCLC), and NCT01496742 (non-squamous NSCLC). Patients’ characteristics are summarized in Table For the analysis of patient-matched CTCs (ISET platform) and tumors, blood and tumor tissue samples were obtained from 106 newly diagnosed patients with histologically confirmed NSCLC and advanced-stage disease; these patients were treated at the Pneumology Department,  between January 2008 to December 2013. After CTC assessment, we selected 80 patients positive for CTCs for further analysis (see below); 20 patients presented initially with metastatic disease, and 60 patients presented with early-stage, but developed metastasis during treatment or follow-up. The main clinical and histopathological parameters of the 80 patients included in the study are summarized in Table Automated IHC staining was carried out according to the manufacturer's protocol on the BenchMark XT platform . IHC evaluation was performed using CONFIRM anti-Total c-MET (SP44) rabbit monoclonal primary antibody  and the ultraView Universal DAB Detection Kit . Primary antibody was incubated for 16 minutes at 37°C. The MET IHC scoring was based on a combined assessment of membranous and cytoplasmic expression in tumor cells, and was evaluated blindly by three pathologists , with an overall concordance of 96%, using a qualitative 4-level intensity scale (0–3). Tumor cells showing MET protein intensities of 2 and 3 were considered positive. Sample status was determined using the Onartuzumab scoring criteria in lung: positivity was defined as having ≥ 50% of tumor cells positive for membranous and/or cytoplasmic MET immunostaining . In addi® Circulating Tumor Cell Kit, as per manufacturer's instructions . CTC were identified following the CellSearch guidelines, and CTC enumeration was expressed as the number of CTCs detected in 7.5 ml blood. For the evaluation of MET expression, Alexa 488-conjugated MET antibodies  were added to the open 4th antibody position of the CellSearch system for automated MET staining. Normal blood samples spiked with control cell-lines that express high (EBC-1), medium (HCC1954) and low levels of MET (HCC70) were used as scoring controls for the quantification of MET expression in patients’ samples , and only samples received before 96 hours from collection were analyzed . Upon receipt, samples were processed for CTCs enumeration on the CellSearch platform using the CELLSEARCH samples . The levCTC enrichment by ISET  was performed as per manufacturer's instructions. Briefly, 10 mL of blood were processed though the ISET filter within one hour of blood collection. The ISET filter contains 10 filter-spots, each representing the filtration of 1 ml blood. May-Grunwald-Giemsa (MGG) staining was performed on 4 filter spots, and these filter spots were examined for the presence of circulating non-hematological cells with malignant (CNHC-MF), uncertain (CNHC-UMF) and benign features (CNHC-BF), as previously reported , 33. Sam2 analysis or the Mann-Whitney test when applicable. For outcome analysis, the primary end-point was progression-free survival (PFS) calculated from the time of histological diagnosis to the time of progression or death. Patients who were still alive at the end of study were censored at the end of study date. PFS was calculated using Kaplan-Meier method and a survival plot was generated.Concordance between tumor status and MET expression in CTCs was determined by Spearman rank correlation. Correlation coefficient ≥ 0.70 was considered good concordance. MET expression in tumor tissue and CTCs, analyzed as a binary positive vs. negative variable, was compared with clinico-pathological variables such as age, gender, smoking status, and tumor size, by using the χ


Curing, though important is sometimes underrated in concrete production. This dataset provided shows the effects of four (4) different methods of curing on two distinct mix ratios. The data provided in this article are for a study that was conducted on one hundred and sixty (160) cube samples of mix ratios 1:2:4 and 1:1.5:3 while employing four (4) different methods of curing. The data given in the article displays the finding of the study. The findings can aid in prediction and optimization of concrete behavior and compressive strength when any of the curing methods are utilized. Specifications TableValue of the data•This data is valuable because concrete is a widely used material and relevant information on its uses can be very significant.•The data presented shows the effects of the different curing methods on the strength of concrete.•The data may be relevant in the development of standards or codes of practice for concrete production in rural and arid areas.•The data presented can be used to investigate the effects of mixing and curing methods on concrete of different mix proportions.•The data presented can be used to develop an optimum method for concrete production.1The data provided is focused on the effects of curing on the compressive strength of concrete. Concrete is a universally accepted construction material 2The materials for this research were Ordinary Portland Cement (OPC) which was used as the binder throughout the research, fine, and coarse aggregates. The coarse aggregate size used 20 mm diameter, the fine aggregates used were graded appropriately. 160 concrete cube specimens were cast of dimensions 150 mm × 150 mm × 150 mm in steel mould in accordance to


HGF/c-Met system has covered, in the past two decades, multiple areas of research, from basic cell biology to clinical trials. This research topic witnessed a good deal of scientific interest and progress. These two molecules, in fact, have been discovered as being crucial elements in mediating multiple biological activities such as cell proliferation, survival, motility, morphogenesis and angiogenesis. It has been clearly demonstrated that these pleiotropic and complex biological functions act together during organogenesis and tissue homeostasis; however it can give rise to a broad spectrum of pathologies too, when the HGF/c-Met system is disrupted. Since I consider this topic of wide scientific interest, I felt the need to dedicate a special focus on this particular subject. The articles in this Special Issue are a collection of primary research papers and syntheses that have the merit to illustrate the most topical aspects of the HGF/cMet system biological functions. The goal is to provide a state-of-the-art overview, emphasize the new emerging paradigms and identify the current challenges to further scientific progress.i.e., the relevance of HGF as a modulator of cancer and non-cancerous cells in the neoplastic microenvironment. Interestingly a mathematical model that describes the epithelium-mesenchyme transition of cancer cells has also been reported. In this regard, it is worth highlighting that research areas, having been to date historically separated, are becoming more related with multidisciplinary research teams representing, in my opinion, the future of good science.In this short editorial, I wish just to highlight the most relevant “take-home-messages” of this project. One of the highly topical subjects addressed by several articles concerns the involvement of the HGF/c-Met system in the onset and progression of cancer. This Special Issue contains manuscripts reviewing c-Met-dependent malignant behavior of the cells: the adaptor proteins and kinases involved in this process, and the other tyrosine-kinase receptors that enhance c-Met activation and signaling. Moreover, current knowledge about microRNAs known to regulate c-Met expression in tumors is also reported: this particular subject represents, in my opinion, a new promising field of research for basic science, cancer therapy, and cancer diagnosis. Other authors, highlight another important aspect of cancer progression, The role of the HGF/c-Met system in tissue homeostasis and repair represents another relevant topic that has been outlined in this issue. In particular it has been pointed out that the HGF/c-Met system is involved in muscle regeneration and function, protection from acute tissue injury, chronic fibrosis, and in neurodegenerative diseases. This has led to ongoing HGF-based clinical trials for the treatment of patients with amyotrophic lateral sclerosis and spinal cord injury. Since HGF/c-Met system is a well-known modulator of the harmonic morphogenesis during embryonic development, the ability of HGF to drive tissue repair represents, in my opinion, a crucial link of this system to the success of stem cell-based therapies.Finally, this Special Issue provides basic scientists and clinicians with a comprehensive, up-to-date overview of the newly available biotechnologies and compounds with potential for preventing organ failure and treating cancer.In closing, I wish to thank the other Guest Editors for their personal contributions in the organization of this issue. A special mention is due to Prof. Maria Prat, for her commitment and support, which has been crucial to the success of this Special Issue. Moreover, I wish to personally thank all the authors who contributed and collectively made this dedicated issue possible.


A rare case of IgG4-related disease (IgG4-RD) manifesting as nodular scleritis is presented in a 20-year-old female. Patient complained of left eye pain and redness for one week. Ocular examination together with ancillary testing led to the diagnosis of nodular scleritis. Since the patient did not show apparent improvement after one week of systemic steroidal treatment, she underwent a biopsy of the affected area revealing histopathological characteristics of IgG4-RD. Long-term treatment with corticosteroids and a steroid-sparing agent (methotrexate) led to significant improvement in signs and symptoms. This case highlights the significance of IgG4-RD in the differential diagnosis of scleritis and raises the question as to whether various organs affected by IgG4-RD may have different underlying pathophysiological mechanisms in which pathogenic T cells play a role. Immunoglobulin G4-related disease (IgG4-RD) is an immune-mediated condition that can affect almost any organ. The typical ophthalmic manifestation is lacrimal gland enlargement (dacryoadenitis). The diagnosis can be confirmed by blood tests and histology during the acute phase. We present a unique case of nodular scleritis due to isolated IgG4-RD, which responded well to immunosuppressive and immunomodulatory treatment.A 20-year-old female presented with complaints of discomfort and redness of her left eye of one week's duration. She had no associated visual complaints. Ocular examination revealed a localized inflamed superior bulbar conjunctival swelling in the form of scleritis. Ocular motility was preserved, and visual acuity was 6/6 in both eyes. Anterior segment ocular computerized tomography (OCT) demonstrated a thickened episclera and sclera with hyporeflective areas representing fluid in the region, confirming the diagnosis of scleritis Figures . Blood tThe patient then underwent a biopsy of the affected area to confirm the diagnosis and to rule out conjunctival lymphoma. Histopathological analysis of the specimen revealed an intensive lymphoplasmacytic infiltration with a large number of IgG-positive plasma cells, >40% positive for IgG4. There were >30 IgG4 cells per high-power field. A large number of CD4+ positive cells (~50%) were observed in the specimen Figures . A positRecurring anterior and posterior unilateral scleritis had been reported in a 63-year-old woman with a 13-year history of the disease . HistopaIgG4-RD can occur in any ocular adnexal tissue, for example, lacrimal glands, extraocular muscles, and cavernous sinus . The sigThe biopsy in the case presented here met the criteria for probable IgG4-related inflammatory pseudotumor , which wCD4+ cytotoxic T cells appear to play an important role in disease pathophysiology. These cells have been shown to be dispersed throughout IgG4-RD lesions, as documented in our patient Figures . The cloLong-term glucocorticoid treatment , 3, 5 isBased on these findings, we believe that the various organs affected by IgG4-RD may have different underlying pathophysiological mechanisms in which pathogenic T cell may play a different role.


Aberrant signaling of the hepatocyte growth factor (HGF)/c-Met pathway has been identified as a promoter of tumorigenesis in several tumor types including head and neck squamous cell carcinoma (HNSCC). Despite a relatively low c-Met mutation frequency, overexpression of HGF and its receptor c-Met has been observed in more than 80% of HNSCC tumors, with preclinical and clinical studies linking overexpression with cellular proliferation, invasion, migration, and poor prognosis. c-Met is activated by HGF through a paracrine mechanism to promote cellular morphogenesis enabling cells to acquire mesenchymal phenotypes in part through the epithelial-mesenchymal transition, contributing to metastasis. The HGF/c-Met pathway may also act as a resistance mechanism against epidermal growth factor receptor (EGFR) inhibition in advanced HNSCC. Furthermore, with the identification of a biologically distinct subset of HNSCC tumors acquired from human papillomavirus (HPV) infection that generally portends a good prognosis, high expression of HGF or c-Met in HPV-negative tumors has been associated with worse prognosis. Dysregulated HGF/c-Met signaling results in an aggressive HNSCC phenotype which has led to clinical investigations for targeted inhibition of this pathway. In this review, HGF/c-Met signaling, pathway alterations, associations with clinical outcomes, and preclinical and clinical therapeutic strategies for targeting HGF/c-Met signaling in HNSCC are discussed. Carcinomas of the head and neck account for an increasing number of cancer cases worldwide with an incidence of more than 500,000 new cases and 380,000 deaths annually . Head anThe current FDA-approved agents for the treatment of HNSCC include methotrexate, 5-fluorouracil, bleomycin, cisplatin, cetuximab, docetaxel, and most recently pembrolizumab and nivolumab. Unfortunately, despite the multitude of therapeutic agents and treatment modalities including surgical resection and radiation, the five-year survival rate for HNSCC has failed to improve over the past few decades with dismal response rates in recurrent/metastatic (R/M) patients ,9. AmongThe discovery of overexpression of HGF and its receptor c-Met have been reported in a majority of HNSCC tumors, precipitating multiple investigations into the tumorigenic effects of this pathway in HNSCC . ActivatHGF is a pleiotropic plasminogen-like cytokine cytogenically located at 7q21.11 and originally discovered for its mitogenic effects on parenchymal hepatocytes . IdenticIn HNSCC, the majority of HGF is secreted by tumor-associated fibroblasts (TAFs) in the tumor microenvironment as an inactive zymogen, requiring proteolytic cleavage by the membrane-bound protease matriptase  33]. Cl. Cl33]. MET located on the long arm of chromosome 7 at position 7q31.2 [Identified as the sole receptor for the HGF ligand, c-Met is a receptor tyrosine kinase encoded by the proto-oncogene n 7q31.2 . The c-Mn 7q31.2 ,36. HGF n 7q31.2 ,38. Subsn 7q31.2 ,39. Phosn 7q31.2 .MET mutations, amplification of the MET gene, and overexpression of both c-Met and HGF protein. Overexpression of c-Met protein is the most frequently observed alteration presenting in up to 90% of HNSCC tumors, with mRNA overexpression frequently reported as well [MET amplification and increased MET gene copy number are observed at a low frequency in HNSCC tumors, they are associated with the overexpression of c-Met protein [Increased activation of the HGF/c-Met signaling pathway results from a variety of genetic abnormalities including  as well ,40,41,42 protein .MET mutations have been identified in the MET tyrosine kinase domain, sema, and juxtamembrane domains in HNSCC patient tumors. In a study by Di Renzo et al., the activating point mutation Y1235D was detected at a higher incidence in metastatic lymph tissues from HNSCC patients compared to the corresponding primary tissue suggesting clonal selection of the mutation and evidence that c-Met modulates metastasis [MET mutation frequency and identified two novel mutations (T1275I and V1333I) in the tyrosine kinase domain as well as mutations in the sema  and juxtamembrane (T1010I and R988C) domains [MET mutations that result in exon 14 skipping, deletion of the juxtamembrane domain, and loss of Casitas B-lineage lymphoma (Cbl) E3 ubiquitin-ligase recruitment, were only identified in less than 1% of HNSCC tumors, but potentially require more advanced technical methods for increased detection [In addition to c-Met and p-Met overexpression, tastasis . In supptastasis ,46. In a domains . Lastly,etection ,48.HGF gene expression has been reported in up to 58% of R/M HNSCC cases [Paralleling c-Met expression, HGF protein overexpression has been observed in 45% of primary HNSCC tumors as well, while positive CC cases ,41. MarsCC cases ,49. In HCC cases . DysreguCC cases .Aberrant HGF/c-Met signaling in HNSCC promotes tumor progression and enables the development of distant metastasis by increasing the invasive capacity of HNSCC tumor cells. Multiple studies have demonstrated that recombinant HGF stimulation of both primary and metastatic HNSCC cell lines leads to increased migration and invasion ,50,51. SHGF/c-Met signaling stimulates the morphogenesis of epithelial cells to acquire these aggressive motile phenotypes through EMT. During EMT, cells lose their characteristic epithelial phenotype and in turn acquire mesenchymal features including front-rear polarization, an elongated spindle-like morphology, and migratory potential resulting in the metastatic progression of several carcinomas including HNSCC ,54. StudThe HGF/c-Met pathway employs multiple mechanisms to induce cell migration and invasion, and in addition to E-cadherin loss, HGF induces increased expression of matrix metalloproteinases (MMPs) in HNSCC. MMPs are a class of proteases identified in tumor cell metastases and are responsible for the degradation and remodeling of the extracellular matrix (ECM) . In primTP53-induced glycolysis and apoptosis regulator (TIGAR). Targeted inhibition of c-Met was found to significantly downregulate expression of TIGAR in nasopharyngeal cells in turn reducing intracellular nicotinamide adenine dinucleotide phosphate (NADPH) levels necessary for evading apoptosis [Another mechanism by which HGF/c-Met signaling promotes tumor progression and cell survival is through the p53 protein product poptosis . Subsequpoptosis ,64. ThisHGF further supports HNSCC tumor development through the promotion of angiogenesis. A hallmark of cancer progression, angiogenesis is stimulated by HGF-induced production of well-established pro-angiogenic factors including interleukin (IL)-8, vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF). Previous reports have correlated increased IL-8 and VEGF levels in HNSCC tumors with larger tumor volume, increased recurrence and shorter disease-free intervals ,66. In vMET gene is typically undetectable in normal lymph tissue, MET expression was found in up to 40% of HNSCC lymph nodes assessed, 24% of which were histopathologically confirmed metastatic [MET mutations are selectively identified in metastatic lymph, and not in the primary tissue, confirm a role of the HGF/c-Met pathway in metastasis [Alterations in HGF/c-Met signaling in HNSCC therefore unsurprisingly correlate with increased incidence of regional and distant metastases. While distant metastasis rates remain low, one of the most significant prognostic indicators for HNSCC patients is the presence of metastatic disease in the cervical lymph nodes, correlating with a 50% decrease in survival ,71. Sevetastatic . This datastasis .MET mutations or c-Met expression and clinicopathological outcomes [Studies comparing c-Met expression with clincopathological parameters consistently report positive and overexpressed c-Met protein in HNSCC tumor samples ,76,77,78outcomes . DespiteHGF, both tumoral and peripheral, has also been evaluated as a potential biomarker for the progression of HNSCC . In addiMET gene amplification was found (3%), and regardless of HPV status a statistically significant correlation between prognosis and either MET amplification or c-Met protein expression was not observed [Potential explanations for the variability in these results may stem from the heterogeneity and diverse tumor sites of the HNSCCs evaluated. Furthermore, analytical parameters and cut-offs for high versus low expression levels, as well as sample size, were not consistent across all studies. Finally, confounding clinical variables, such as HPV status, were not always included in the statistical analysis. HPV is an increasingly prevalent risk factor for the development of HNSCC, with the rise in oropharyngeal cancers in the U.S. in part attributed to increased incidence of HPV infections . Interesobserved . In contobserved . DespiteEGFR gene copy number and protein expression in patient tumors have not been found to be predictive of cetuximab response [The EGFR-directed monoclonal antibody, cetuximab, is one of three targeted agents approved to date by the FDA for the treatment of HNSCC, and improves survival when added to front line platinum . Despiteresponse ,93, a weresponse ,94,95, tKRAS wild-type colorectal cancer [Preclinically, the EGFR ligand TGF-α stimulated activation of c-Met in HNSCC cell lines, through prolonged tyrosine phosphorylation and increased c-Met protein expression . Moreovel cancer  and lungl cancer ,98,99, bl cancer . c-Met il cancer .Together, these data suggest that HGF/c-Met pathway inhibition may overcome resistance to anti-EGFR therapy in R/M HNSCC, such as in patients with clinical cetuximab resistance. Given the cross-talk and mutual compensation between the HGF/c-Met and EGFR signaling pathways, optimal benefit may be achieved by continuing EGFR blockade concurrent with HGF/c-Met pathway inhibition, in spite of established cetuximab clinical resistance.Several agents have been developed to target both the HGF ligand and c-Met receptor including small molecule tyrosine kinase inhibitors (TKIs), MAbs, and competitive HGF antagonists and c-Met receptor decoys . Crizoti50 < 0.5 μM) [Other c-Met TKIs that have undergone preclinical investigation in HNSCC include foretinib, tivantinib, and SU11274. Foretinib (GSK1363089) is a multikinase inhibitor predominately targeting c-Met and VEGF receptor-2 . When te 0.5 μM) . However 0.5 μM) . Tivanti 0.5 μM) . A recen 0.5 μM) . However 0.5 μM) . Preclin 0.5 μM) ,41. Howe 0.5 μM) .Several anti–HGF antibodies have been developed for the inhibition of c-Met mediated neoplastic effects as well. Ficlatuzumab (AV-299) is an IgG1 HGF MAb shown to selectively bind and inhibit HGF activity. After successful preclinical assessment in NSCLC, and multiple phase I clinical trials establishing a safety profile, ficlatuzumab was also evaluated in HNSCC . Kumar eOther targeted agents in development for selective inhibition of HGF/c-Met signaling include Onartuzumab (MetMab), Emibetuzumab (LY2875358), and NK4. Onartuzumab is a humanized monoclonal anti-c-Met antibody designed to bind to the receptor and inhibit ligand-induced activation of the HGF/c-Met pathway . Proven Preclinical evaluations of these agents targeted against the HGF/c-Met pathway in HNSCC predominately begin with monolayer 2-D cell culture models assessing drug efficacy, cytotoxicity, and influence on cell viability. Fortunately, a multitude of diverse HNSCC cell lines have been established allowing for a more comprehensive investigation of disease pathogenesis and druggable targets . HoweverTP53 and PI3KCA [Xenograft models continue to provide an effective means for identifying inhibitors for further clinical development . Howeverd PI3KCA ,130. FurGiven that the HGF/c-MET pathway is involved in multiple stages of HNSCC tumor progression, it is a highly promising therapeutic target for this disease. As discussed, many types of inhibitors have been developed to abrogate activation and signaling of this pathway, and several of these HGF/c-Met targeting agents have been evaluated in completed or on-going clinical trials for HNSCC patients as summarized in The first report to evaluate a c-Met inhibitor in HNSCC was a phase II study which tested the efficacy, safety, and tolerability of single-agent foretinib, an ATP-competitive inhibitor of c-Met and VEGFR2, in patients with R/M HNSCC . While tPreclinical studies suggest that combination approaches with HGF/c-Met inhibition may be promising. Two subsequent studies with c-Met TKIs in combination with cetuximab are ongoing (Studies using HGF directed monoclonal antibodies are limited. One trial using the HGF monoclonal antibody ficlatuzumab, and one in combination with cetuximab is ongoing (Head and neck cancer remains a prevalent disease in need of novel and efficacious treatment strategies for patients. Integral in the development of an invasive, proliferative, and metastatic phenotype, the HGF/c-Met pathway presents as an attractive target for molecular inhibition. Several therapies selectively targeting either c-Met or its ligand HGF have revealed potent antineoplastic effects in preclinical HNSCC models, with some agents advancing to clinical development. While clinical trials for these targeted therapies are still ongoing, inhibition of this pathway along with standardized evaluation of HGF and c-Met expression profiles with clinical outcomes is warranted in order to provide certain HNSCC patients another viable treatment strategy.


Seed-based analysis was performed focusing on triple-network models and six striatal networks. Neurobehavioral scores served as the major outcome factors. The role of seed or peak cluster volumes, or a covariance strength showing Met-carriers > Val-homozygotes were tested for the effect on dopamine. Clinically, the Met-carriers had higher mental manipulation and hallucination scores than the Val-homozygotes. The volume-score correlations suggested the significance of the putaminal seed in the Met-carriers and caudate seed in the Val-homozygotes. Only the dorsal-rostral and dorsal-caudal putamen interconnected peak clusters showed covariance strength interactions , and the peak clusters also correlated with the neurobehavioral scores. Although the triple-network model is important for a diagnosis of Alzheimer’s disease, our results validated the role of the dorsal-putaminal-anchored network by the catechol-O-methyltransferase Val158Met polymorphism in predicting the severity of cognitive and behavior in subjects with Alzheimer’s disease.The catechol-O-methyltransferase enzyme metabolizes dopamine in the prefrontal axis, and its genetic polymorphism  is a known determinant of dopamine signaling. In this study, we investigated the possible structural covariance networks that may be modulated by this functional polymorphism in patients with Alzheimer’s disease. Structural covariance networks were constructed by 3D T1 magnetic resonance imaging. The patients were divided into two groups: Met-carriers (The online version of this article (doi:10.1007/s12035-017-0668-2) contains supplementary material, which is available to authorized users. Dopamine pathways modulate learning, memory, and neuropsychiatric presentations, and the catechol-O-methyltransferase (COMT) gene has been implicated in the enzymatic degradation of dopamine. In humans, a functional single nucleotide polymorphism (rs4680 G to A), consisting of a change in the coding exon at position 158 , has been reported to result in a two- to fourfold decrease in the activity of the COMT enzyme. Consequently, the low enzyme activity in Met/Met homozygotes results in increased dopamine levels that may preferentially affect prefrontal-related tasks [Although the hypothesis of dopamine dysfunction in the early stage of Alzheimer’s disease (AD) is still under debate, several experimental models support the role of Aβ oligomers and dopamine dysfunction . In addiThe dopamine pathway represents one of the major biochemical signals in the striatum. Classically regarded as a motor structure, the striatum subserves a wide range of functions including cognitive, motivational, and emotional processes. In recent years, researchers have started to conceptualize the functional connectivity of distinct neural circuits associated with different sub-regions of the striatum. For instance, the reward-related function has been attributed to the ventral versus superior striatum . SimilarIn 2006, Postuma and Dagher  proposedRecent research has suggested that highly related regions show covariance in morphometric characteristics, the so-called structural covariance. With careful control of confounding factors, structural covariance networks (SCNs) have been used to test the influence of genotypes . The triDopamine may affect cognition by facilitating neuronal synchrony; however, a direct correlation between COMT genotypes and prefrontal-striatal dopamine levels or gene-cognitive profiles remains controversial , 29. TheThe distribution of the genotypes of the Val158Met polymorphism in the patients with AD was in Hardy-Weinberg equilibrium. There were no significant differences in gender, age, and educational level between the two genotype groups  with a cluster size >100 voxels). In the striatal model, the dorsal caudal putamen (DCP) seed had a significantly higher volume in the Val-homozygotes  volume of the Met-carriers and Val-homozygotes using voxel-based morphometry  showed nFor each seed, we explored the genotypic interactions with regard to the topography showing differences in structural covariance strength between seed and peak clusters Fig. D. The leWe also explored whether the seed volumes were correlated with the cognitive test scores in each group  were tested for their effect on dopamine. As more statistically significant neurobehavioral domains were found in the Met-carriers in the network showing higher covariance strength  total score, executive function test, and attention scores in both genotype groups suggest the clinical role of the ventral rostral putamen seed.None of the peak clusters connected to the ventral striatum seed showed greater structural covariance strength in the Met-carriers. This may be due to a minor genotype modulation effect on the ventral striatum-interconnected clusters. The correlations between ventral striatum seed and neurobehavioral symptoms were still significant and displayed a parallel correlation pattern in the superior ventral striatum or inferior ventral striatum, especially in the Val-homozygotes. These results may be related to the identical structural projection zone of the superior ventral striatum –41 and iFor the dorsal caudate nucleus, the anatomical connection has been associated with the dorsolateral prefrontal cortex and executive control regions –48. The Individuals with the Val/Val genotype have been reported to have higher levels of thyroxylase mRNA in mesencephalic dopamine neuronal populations that project to the striatum , which mIn addition to the physiological role of striatal networks, several factors in AD may also contribute to the inconsistent effect of COMT. For example, the genetic expression of COMT and the effect of the dopamine system can be influenced by aging, amyloid load, and disease severity. During the physiological aging process, decreased dopamine release, decreased receptor expression , and reduced transporter expression are found in the caudate, putamen, hippocampus, and prefrontal cortex of human brains , 50. In The striatum network in AD is mediated by different pathways and neurotransmitters, and dopaminergic pathways such as nigrostriatal pathways (substantia nigra and striatum) modulate voluntary movement and mesocorticolimbic pathways  modulate cognitive-behavior-reward function. Although dopamine levels have been reported to be higher in the striatum of individuals with the Val/Val genotype than in those with the Val/Met genotype , the areAn important limitation of this study is that we did not include a control group. The enrolment of controls may have helped to elucidate whether the COMT polymorphism has a similar effect on the normative brain network. Our results support published data on elderly healthy subjects that genetic variations of the COMT polymorphism may mediate pre-frontal-related tasks . HoweverIn AD, the COMT Val158Met polymorphism modulates the striatal network rather than the triple network with regard to predicting symptoms. The genotype group itself, seed volume, or striatal network provided variable predictions of the clinical features. In the striatal network, greater covariance strength in the Met-carriers was found in the DCP- and DRP-interconnected networks that were suggestive of a long-term dopamine-related effect. Along with the significant clinical correlations, the DCP- and DRP-interconnected networks may be considered to be the major networks modulated by the COMT genotype.This study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board of Chang Gung Memorial Hospital. The study participants were treated at the Cognition and Aging Center, Department of General Neurology, Kaohsiung Chang Gung Memorial Hospital. A total of 192 subjects  were included after the consensus of a panel composed of neurologists, neuropsychologists, neuroradiologists, and experts in nuclear medicine . AD was n = 91) and Val-homozygotes (n = 101). The working scheme was as follows. First, the SCNs were established by seed-based correlation analysis. Differences in each seed regional volume were compared between two genotype groups and correlated with the neurobehavioral scores. In order to evaluate the dopaminergic network effects, only the peak clusters showing Met-carriers > Val/Val in covariance strength were considered as statistically significant. The volumes of the significant peak clusters were selected and correlated with cognitive test scores to evaluate the clinical relevance in each genotype group.Because of the limited number of subjects in the Met/Met group, we grouped the Val/Met and Met/Met subjects into the Met-carrier group in all subsequent analysis: Met-carriers  , while tNlaIII restriction fragment length polymorphism analyzed on 10% polyacrylamide gel. Partial digestion and contamination amplification were ruled out by the complete digestion of an intrinsic restriction site and a blank sample in each batch of experiments, respectively. The ApoE genotype was determined using a PCR-restriction fragment length polymorphism assay and restriction enzyme HhaI. ApoE4 carriers were defined as those with one or two E4 alleles.Genotyping of COMT Val158Met was performed using the polymerase chain reaction-restriction fragment length polymorphism method. In brief, a DNA fragment containing the Val/Met polymorphism in COMT was amplified by polymerase chain reaction with primers reported by Lachman et al. [3.MR images were acquired using a 3.0T MRI scanner . Structural images were acquired for SCN constructions using the following protocols: a T1-weighted, inversion-recovery-prepared, three-dimensional, gradient-recalled acquisition in a steady-state sequence with a repetition time/echo time/inversion time of 8600 ms/minimal/450 ms, a 256 × 256 mm field of view, and a 1-mm slice sagittal thickness with a resolution of 0.5 × 0.5 × 1 mmhttp://www.fil.ion.ucl.ac.uk/spm/). The T1 images were reoriented, realigned, and normalized using the standard Montreal Neurological Institute space. The images were then segmented into GM and white matter. Related tissue segments were used to create a custom template using the diffeomorphic anatomical registration using exponentiated lie algebra (DARTEL) approach. The DARTEL approach is one of the highest ranking registration methods in patients with AD [Image preprocessing and statistical analysis were performed using SPM8  with a cluster size >100 voxels.From the modified GM images, the GM volumes of a 4-mm radius sphere around the seed coordinates were also calculated, followed by 10 separate correlation analyses using the extracted GM volumes as the covariates of interest, to form the SCN. The two genotype groups were modeled separately. Based on the equivalent sample sizes in each genotype group, T contrasts were used to identify voxels that showed positive correlations for each seed. The results reflected the SCNs anchored by each seed. The threshold was set at In addition, to investigate how genetic variance may interfere with SCN covariance strength, voxels showing significant differences in the regression slopes in each seed-peak cluster correlations were compared that pointed to interactions between Met-carriers > Val-homozygotes. Specific T contrasts were established to map the voxels that expressed significant between-group associations.p < 0.05 with multiple corrections.For the peak clusters showing significant between-group differences, a 4-mm radius sphere was placed on the peak voxel, and the GM volumes were then calculated. To evaluate the clinical significance of the seed or the identified peak voxel, we used correlation analysis with the cognitive test or NPI scores as outcome measures. The threshold was set at t test was used to compare the continuous variables and chi-square test for category variables. Pearson’s correlation was used to analyze the seed or cluster volume on predicting the cognitive or NPI scores. All statistical analyses were conducted using SPSS software . Statistical significance was set at p < 0.05.Clinical and laboratory data were expressed as mean ± standard deviation. The Student Supplementary Figure 1a) four target seeds, (b) seed volume comparisons, and (c) structural covariance networks  in all patients with Alzheimer’s disease with the catechol-O-methyltransferase Val158Met polymorphism . A significantly lower posterior cingulate cortex gray matter seed volume was found in the Met-carriers (p < 0.05). The images were displayed on a standard brain render. (GIF 455 kb)Statistical maps depicting brain areas in which the gray matter intensity covaried with (High resolution image (TIFF 645 kb)Supplementary Table 1(DOCX 21 kb)Supplementary Table 2(DOCX 23 kb)Supplementary Table 3(DOCX 20 kb)Supplementary Table 4(DOCX 19 kb)Supplementary Table 5(DOCX 19 kb)Supplementary Table 6(DOCX 19 kb)Supplementary Table 7(DOCX 18 kb)Supplementary Table 8(DOCX 21 kb)Supplementary Table 9(DOCX 21 kb)Supplementary Table 10(DOCX 20 kb)Supplementary Table 11(DOCX 19 kb)Supplementary Table 12(DOCX 20 kb)Supplementary Table 13(DOCX 20 kb)Supplementary Table 14(DOCX 21 kb)Supplementary Table 15(DOCX 21 kb)Supplementary Table 16(DOCX 22 kb)Supplementary Table 17(DOCX 20 kb)Supplementary Table 18(DOCX 22 kb)Supplementary Table 19(DOCX 20 kb)Supplementary Table 20(DOCX 21 kb)Supplementary Table 21(DOCX 23 kb)Supplementary Table 22(DOCX 25 kb)