Nickel-Titanium (Ni-Ti) alloys are very difficult-to-machine materials causing notable manufacturing problems due to their unique mechanical properties, including superelasticity, high ductility, and severe strain-hardening. In this framework, the aim of this paper is to assess the machinability of Ni-Ti alloys with reference to turning processes in order to realize a reliable and robust in-process identification of machinability conditions. An on-line sensor monitoring procedure based on the acquisition of vibration signals was implemented during the experimental turning tests. The detected vibration sensorial data were processed through an advanced signal processing method in time-frequency domain based on wavelet packet transform (WPT). The extracted sensorial features were used to construct WPT pattern feature vectors to send as input to suitably configured neural networks (NNs) for cognitive pattern recognition in order to evaluate the correlation between input sensorial information and output machinability conditions. Nickel-Titanium (Ni-Ti) alloys are recognized for their excellent electrical, mechanical and damping properties, including superelasticity and shape-memory. Such attributes make these alloys a promising material for a number of applications in different fields like automotive, aerospace and robotics ,2,3.For instance, as Ni-Ti alloys possess high strength, strong corrosion resistance as well as excellent thermal fatigue properties and thermal stability, they have been extensively applied for engine parts production, such as aircraft engine compressor disks, turbine disks, bearing rings, turbine blades and other parts working at high temperature .More recently, Ni-Ti intermetallic compounds, also known with the name of Nitinol, have been introduced in the biomedical sector for the fabrication of coronary stents and orthodontics and orthopedic implants due to the high biocompatibility of the metal alloy ,6.However, due to the high temperatures and stresses generated during machining of Ni-Ti alloys, the latter are classified as difficult-to-machine materials. A rapid tool failure and poor surface quality of the workpiece are generated during Ni-Ti alloys machining due to excessive burr formation, adhesions on the machined surface and microstructure alterations of the workpiece material . MoreoveDiverse research studies discussed the challenges related to machining of Ni-Ti alloys, focusing on process parameters optimization, tool wear identification or workpiece surface quality.The study in  focused Tool wear behaviour and cutting forces in machining of Ni-Ti shape memory alloys under various machining conditions  and different cutting speeds were investigated in . The research study in  demonstrThe machinability of Ni-Ti based shape memory alloys with reference to turning and drilling processes was examined in  by varyiTurning of Ni-Ti alloys was also studied in , where tAn approach to improve the machinability of Ni-Ti alloys through the application of chilled air was studied in , showingAn overview of the machinability of aerospace engine materials with emphasis on titanium and nickel-based alloys was presented in . The enhTo improve and optimize machining processes, a number of studies in the last decades proposed the use of on-line sensor systems for monitoring of tool conditions ,18,19, mTo exploit the sensor monitoring results as a support to decision making systems for machining processes, the development and implementation of advanced sensor monitoring procedures, based on innovative technologies and approaches, is essential.detection of machining process variables through sensorial perception methods;sensor signals processing;relevant sensorial features extraction and selection;implementation of decision making paradigms for diagnosis on machining process conditions;activation of corrective actions.The sequence of activities to be performed in sensor monitoring of machining process conditions can be summarised as follows :detectiUsually, the sensor signals obtained during process monitoring are subjected to signal conditioning  and processing with the aim to generate functional signal features ,27,28,29The signal features extracted through these methods are then supplied to and evaluated by cognitive decision-making support systems based on approaches such as neural networks, fuzzy logic, genetic algorithms, etc. ,35 for tIn this framework, the aim of this paper is to develop an advanced sensor monitoring procedure for turning of Ni-Ti alloys in order to realize a reliable and robust classification of the process quality in terms of machinability conditions.The sensor monitoring procedure implemented during experimental turning tests on Nitinol bars is based on the on-line acquisition of vibration sensor signals, that include valuable information on the machining process.Specifically, vibrations occurring during metal cutting can be classified into dependent and independent of the cutting process. The most renowned type of vibration in machining is chatter, i.e., self-excited vibration, which is detrimental to surface finish and tool life. Vibrations dependent on metal cutting can exhibit specific characteristics as a function of the process. In particular, tool engagement conditions during machining play a significant role in the vibration produced ,36.Due to the high correlation between produced vibrations and machining process characteristics, vibration signals have been successfully employed in the literature for tool condition monitoring applications and in-process prediction of surface roughness during turning processes ,38,39.Accordingly, with the aim to perform process monitoring in turning of Ni-Ti alloys, a vibration sensor system has been selected in this research work to acquire valuable sensor signals that can be correlated to the process conditions.The detected vibration signals are processed through an advanced signal processing method in the time-frequency domain based on wavelet packet transform (WPT) ,31. The c = 40, 55, 75, 100, 130 m/min; feed rate, f = 0.10, 0.15, 0.20 mm/rev; depth of cut, ap = 0.5 mm; coolant: KSM 950 . The depth of cut, ap, was always equal to 0.5 mm, while cutting speed, vc, and feed, f, were varied among the tests. The experimental turning test programme with the relevant cutting parameters is reported in c, f and ap), a total of 15 turning tests were performed.The experimental tests of Ni-Ti alloy turning for machinability assessment were performed on Nitinol  bars of 40 mm diameter and 200 mm length. The selected cutting tools were coated carbide inserts, Kendex TPGN160308, with PVD TiAlN coating (KC5010 grade), rake angle: γ = 0°, clearance angle: α = 11°, edge length L10 = 16.5 mm, thickness S = 3.18 mm, nose angle = 60°, nose radius = 0.8 mm and no chip breaker. Different process conditions were tested using the following parameters: cutting speed, vx, ay, and az along the x, y, and z directions , Italy) was utilized to acquire the sensor signals relative to the 3 acceleration components arections . The 3-aThe acquired vibration signals were processed through advanced methods of signal processing for feature extraction and cognitive pattern recognition with the aim to identify the correlations between sensorial data and process conditions during turning of Nitinol ,29. x, ay, and az detected by the wireless vibration sensor were symmetric about their average value which was differently shifted for each vibration acceleration component with respect to 0 m/s2. This phenomenon is due to the influence exerted on the vibration acceleration components by the gravity acceleration along the x, y and z axes of the vibration sensor, which varies depending on the sensor orientation (their sum is 9.81 m/s2). Initially, before signal segmentation, a pre-processing procedure was applied to remove the offset of each acquired signal with respect to the zero axis. This offset was calculated for each vibration acceleration component as the signal average before the actual machining start; such average was then subtracted to the corresponding signal. Then, the signals were processed by cutting off the signal portions corresponding to the transient conditions . x, ay, and az vibration acceleration signals for the turning test carried out with vc = 40 m/min, f = 0.10 mm/rev, ap = 0.5 mm, where the signal portion to be considered for further analysis  is delimited by vertical red lines. An automatic cut off procedure was carried out by calculating the square of each signal and using an experimentally determined threshold  to identify the beginning and end of the signal relative to actual machining.The three vibration acceleration components ax, ay, and az component.Afterwards, all the cut signals were reduced to a common minimum length  to obtain the pre-processed vibration acceleration signals. The latter signals were subdivided into five equal parts of 10,000 samples for further signal analysis, obtaining a total of 75 vibration acceleration signal specimens for each aFrom the pre-processed vibration acceleration signal specimens, features capable of adequately describing the signal and maintaining the relevant information about the process were extracted utilizing the wavelet packet transform (WPT) method in the time-frequency domain .Generally, the basis function of wavelet transform (WT) are small waves (wavelets) of varying frequency and limited duration; in this way, the signal is represented as a superposition of wavelets. Therefore, the WT can extract information in the time domain with reference to different frequency bands. This simultaneous time-frequency decomposition gives the WT a special advantage over the traditional Fourier transform in analysing non-stationary signals ,32,33.The WPT is a generalization of wavelet decomposition that provides level by level transformation of a signal from the time domain into the frequency domain. In the decomposition procedure, high-pass and low-pass filters are utilized to decompose an incoming signal S . At the x, ay, and az vibration acceleration components was realized using the Daubechies db3 mother wavelet. The decomposition procedure, performed separately for ax, ay, and az, was carried out up to the third level, generating a total of 14 wavelet packets of coefficients for each vibration acceleration component: for each packet, five statistical features, i.e., mean, variance, skewness, kurtosis, and energy, were calculated  of the ax is illustrated in x, ay, and az, were obtained.As an example, the WPT feature extraction procedure for packet A of aWith the scope to assess the machinability corresponding to each turning test, three process quality parameters were taken into account: crater wear, flank wear, and machine vibrations level during machining. These three process quality parameters were ranked within a grading scale between 1 (good) and 5 (bad) on the basis of the expert knowledge of a skilled turning operator. In particular, the machine vibrations level was evaluated by an expert operator who carried out his assessment on the basis of his experience as well as with the support of a portable vibration measurement instrumentation. The flank wear and crater wear were categorized by the operator into the five different wear levels by taking into account as critical flank wear value VBmax = 0.6 mm indicated by the standard on tool life testing with single-point turning tool (ISO 3685:1993), and as critical crater wear value the crater front distance KF = 0.02 mm. The machinability was then classified as Acceptable or Poor according to the following rule: in case a ranking ≥ 4 was verified for any of the three quality parameters, then the machinability was classified as Poor. In all the other cases, it was considered Acceptable . The macThe obtained WPT pattern feature vectors were utilised as input to diverse neural networks (NN) for cognitive pattern recognition  aimed at® environment [-input layer with five nodes, according to the number of input feature vector elements ;-hidden layer with a number of nodes equal to 5, 10 or 15;-output layer with only one node, providing a binary target value associated to machinability condition: 0 = Acceptable; 1 = Poor.The NN implementation was carried out in the MatLabironment  using th−7, and the maximum mu parameter value was 1 × 1010. The NN training stops as soon as one of these conditions occurs: the maximum number of epochs is reached, the performance is minimized to the goal, the performance gradient falls below the selected minimum, and mu exceeds its maximum.The NN training set was built up by mating the correct binary target value to each of the 75 vibration acceleration signal specimens in order to map the input pattern feature vector to the output machinability condition: the 75 obtained training cases included 45 Acceptable and 30 Poor machinability cases. The NN training function was based on Levenberg–Marquardt algorithm with the following training parameters: the maximum number of the epochs was set at 1000, the performance goal was fixed to 0, the minimum performance gradient was equal to 1 × 10a − Od), where Oa is the actual output and Od the desired output. The NN classification is considered correct if −0.5 ≤ E ≤ +0.5. The ratio of correct classifications over the total training cases yields the NN success rate (SR).The leave-k-out method (k = 1) was employed: one by one, each pattern feature vector was removed in turn from the training set for testing and the remaining patterns were used for training . The sucx, ay, and az, with reference to the diverse NN configurations  and all the 14 WPT pattern feature vectors. The NN SR was reported for the three diverse types of machinability classification: SR for the identification of Acceptable cases, SR for the identification of Poor cases, and overall SR corresponding to all cases (Acceptable + Poor).The NN performance expressed as SR (%) are shown in x component ranges between 77.8–93.3% in the case of Acceptable machinability identification, 50.0–83.3% in the case of Poor machinability identification, and 68.0–88.0% for Overall cases. The best NN SR value (93.3%) obtained in the identification of Acceptable machinability conditions was obtained with the 5-5-1 NN configuration and the ADD WPT packet, while the best NN SR value (83.3%) is achieved for Poor machinability conditions with the same NN configuration (5-5-1) and WPT packet (DAD). The best NN SR value (88.0%) for Overall  machinability classification is obtained for the 5-5-1 NN configuration and the DAD WPT packet showing a good balance between the identification of Acceptable (SR = 91.1%) and Poor (SR = 83.3%) machinability conditions.y vibration acceleration component  in terms of Overall classification is obtained for two NN configurations: 5-10-1 and 5-15-1. For the 5-10-1 NN, the WPT packet that performs best is the ADA, giving a SR = 93.3% for Acceptable machinability and a SR = 80.0% for Poor machinability. For the 5-15-1 NN, the best performing WPT packets are the DA, giving a SR = 95.6% for Acceptable machinability and a SR = 76.7% for Poor machinability, and the DD with SR = 93.3% for Acceptable machinability and SR = 80.0% for Poor machinability. Thus, the 5-15-1 NN displays a better performance in the identification of Poor machinability conditions using the DD than when using the DA packet.Summarizing, x, and az display the same behaviour with a maximum Overall SR equal to 88.0%. However, ax exhibits a better balance in the identification of Acceptable and Poor machinability.In x, ay, and az were detected through a sensor monitoring system endowed with a three-axis wireless vibration sensor mounted on the tool holder in proximity of the tool insert.An on-line sensor monitoring procedure based on the acquisition of vibration sensor signals during turning of Ni-Ti alloys was implemented to achieve the in-process assessment of machinability conditions. The three vibration acceleration components aAn advanced signal processing method in the time-frequency domain, wavelet packet transform (WPT), was applied to the pre-processed vibration acceleration signals with the aim to extract relevant features able to maintain the relevant information about the process. These extracted features were used to construct WPT pattern feature vectors to feed to suitably configured supervised neural networks (NN) for the identification of machinability conditions.The NN performance was classified in terms of the NN success rate (SR), i.e., the ratio of correct classifications over the total training cases. x and az give the same maximum overall SR = 88.0%, against the ay maximum overall SR = 85.3%. Moreover, ax showed the best balance in the identification of Acceptable (SR = 93.3%) and Poor (SR = 83.3%) machinability conditions.As regards the NN SR for each vibration acceleration component, it was shown that ax, the DDA pattern feature vectors for ay, and the ADA and DD pattern feature vectors for az.As regards the performance of the 14 WPT pattern feature vectors used as input to the NN, the best results were obtained with the DAD pattern feature vectors for ax, ay, and az vibration acceleration components and included together in sensor fusion pattern feature vectors to feed to the NN with enhanced synergical input information.The obtained results showed that the NN SR for Acceptable machinability identification were always higher than for Poor machinability. As shown in


Histologic grade is the most important predictor of the clinical outcome of non-muscle invasive  papillary urothelial carcinoma (NMIPUCa), but its ambiguous criteria diminish its power to predict recurrence/progression for individual patients. We attempted to find an objective and reproducible histologic predictor of NMIPUCa that correlates well with the clinical outcome.A total of 296 PUCas were collected from the Departments of Surgical Pathology of 11 institutions in South Korea. The clinical outcome was grouped into no event (NE), recurrence (R), and progression (P) categories. All 25 histological parameters were numerically redefined. The clinical pathology of each case was reviewed individually by 11 pathologists from 11 institutions based on the 2004 WHO criteria and afterwards blindly evaluated by two participants, based on our proposed parameters. Univariate and multivariate logistic regression analyses were performed using the R software package.The level of mitoses was the most reliable parameter for predicting the clinical outcome. We propose a four-tiered grading system based on mitotic count (> 10/10 high-power fields), nuclear pleomorphism , presence of divergent histology, and capillary proliferation (> 20 capillary lumina per papillary core).The level of mitoses at the initial bladder biopsy and transurethral resection (TUR) specimen appeared to be an independent predictor of the Ta PUCa outcome. Other parameters include the number of mitoses, nuclear pleomorphism, divergent histology, and capillary proliferation within the fibrovascular core. These findings may improve selection of patients for a therapeutic strategy as compared to previous grading systems.The online version of this article (doi:10.1186/s13000-017-0639-y) contains supplementary material, which is available to authorized users. The four existing grading systems  of the urinary bladder, with tumors staged as non-invasive intraepithelial (Ta) or tumors with invasion of the lamina propria/submucosa T1), are known to recur frequently (in up to 70% of cases), and occasionally (in up to 40% of cases) progress , are kno. The samg et al. . In viewg et al. . Many stg et al. .The present study was conducted to identify more objective and reproducible histological predictors that may correlate well with the clinical outcome, and compare these to the previous histological grading systems. Eleven uropathologists evaluated light microscopic histological parameters together in three rounds, using a multihead microscope. Through this study, vigorous attempts were made to select all possible histological parameters as countable variables. Each parameter was evaluated using univariate and multivariate analyses, to determine whether these variables had statistically significant effect in predicting the clinical outcome.Surgically removed NMIPUCs of the urinary bladder were collected from the surgical pathology archives of 11 institutions in South Korea. The inclusion criteria were as follows: (1) pTa or pT1 stage at the initial bladder biopsy; and (2) a 5-year minimum follow-up period for non-event (NE) cases. The exclusion criteria, on the other hand, were as follows: (1) a prior history or the concurrent presence of urothelial carcinoma either in the ureter or in the renal pelvis; and (2) evidence of associated urothelial carcinoma in situ. A total of 296 cases were retrieved . The number of cases contributed by each institute was 95, 47, 38, 22, 21, 21, 17, 15, 14, 4, and 2.< 3 cm vs. > 3 cm. The distribution of each group is shown in Table The retrieved cases were classified into three clinical subgroups: no event (NE), recurrence (R), and progression (P). NE was defined as cases with no evidence of tumor on the follow-up imaging study, urine cytology, or cystoscopy for at least 5-year follow-up duration; R was defined as cases showing a new tumor occurrence with the same or lower stage at least 3 months after the initial resection; and P was defined as a cases showing new tumor development with a higher stage than the initial stage, or metastasis to the lymph nodes or other organs. We collected clinical information on the patients from the medical records, including 1) age, 2) sex, 3) site, number, and size/volume of tumor in first biopsy, 4) interval to the 2nd event, 5) number of recurrences, 6) the type of final operation, 7) survival, 8) cause of death, and 9) site of metastasis. All types of specimens  were included, but were not defined separately. However, there was no partial or radical cystectomy specimen  among the 296 cases. The number of tumors was divided into two groups: single vs. multiple. The tumor size was also divided into two groups: For microscopic examination, hematoxylin and eosin (H&E)-stained glass slides of formalin-fixed, paraffin-embedded tissue of the tumors were retrieved. Interobserver discrepancy had been solved through several round-table multihead microscopic examinations involving 11 pathologists from 11 institutions, during which consensus opinion was reached. Although the proper muscle inclusion was not verified by the reviewers in all samples, 11 contributors had reviewed the original diagnosis and pathological stage, not only by a slide review, but also from the surgical records. The 2nd biopsy was routinely performed 3 months later for check-up of incomplete resection . Even if the initial diagnosis was NMIPUCa , the cases with a short-interval change in the T stage were excluded and were regarded as an inaccurate diagnosis. The clinical pathology of each case was reviewed individually by 11 pathologists from 11 institutions based on the 2004 WHO criteria and was afterward blindly evaluated by two participants, based on our proposed parameters. The histological parameters that were examined are shown in Table http://www.R-project.org/>).All of the aforementioned parameters were evaluated in two paired comparison groups  at each stage. To identify the factors influencing R and/or P, univariate and multivariate logistic regression analyses were performed. To investigate the diagnostic utility of the new grading system, it was compared with the previous grading systems by area under the curve (AUC) of receiver operating characteristics (ROC) curves. All the statistical analyses were performed in the R software package , size , mitotic count , mitotic level , and capillary proliferation in fibrovascular cores  were associated with tumor recurrence. Nuclear pleomorphism showed borderline significance for association with recurrence of PUC-Ta , significant nuclear pleomorphism , presence of divergent histology, and significant capillary proliferation (> 20 capillary lumina per papillary core), the tumors were upgraded: for example, grade 1 became grade 2, grade 2 became grade 3, and grade 3 became grade 4. We designed three similar but slightly different grading schemes.p-value <0.05). However, the differences between them were not statistically significant  , including mitotic level and number of mitoses, level of apoptosis, necrosis, whirling appearance, and capillary proliferation, which had not been evaluated prior to this study, were selected, as well as other histological factors mentioned in the literature. Thereafter, two pathologists blindly evaluated all 296 cases to determine interobserver reproducibility. Some parameters appeared to be influenced by fixation and stain conditions. Therefore, intranuclear groove and nucleolar prominence, which may be produced by procedural artifacts, were considered as low-priority parameters.In the univariate analysis of T1-stage tumors, only a divergent histology correlated with progression. We considered that the pathological stage-factor, with the presence of stromal  invasion, was the most important factor dictating biological behavior from among the histological factors. This finding was in accordance with the WHO recommendation that grading is performed only for noninvasive PUC (PUC-Ta), and with other reports in the literature . TherefoUnlike T1 tumors, Ta tumors had many clinical and histologic parameters that influenced the clinical outcome. Among the clinical factors, the number and size of tumors correlated with recurrence, while patient’s age was associated with progression. In terms of histological factors, mitotic count, mitotic level, and capillary proliferation correlated with recurrence. Cell density, nuclear pleomorphism, hyperchromasia, nuclear groove, prominent nucleoli, necrosis as well as mitotic count and level correlated with progression. Apoptosis and capillary proliferation disclosed borderline significance for progression.It is worth noting that mitotic count showed the highest OR in the prediction of both recurrence and progression of PUC-Ta. In the early twenty-first century, many studies had focused on mitotic index  of Ta/T1 urothelial carcinomas, and have reported those as associated with tumor recurrence –19. HoweThe importance of mitotic count has previously been emphasized for histological grading of NMIPUCa , 23. PicMitotic level has not received much attention either. The upper level of mitosis (level 3 mitosis) correlated with increased mitotic count and worse clinical outcome in our cohort. If a bulky mass is evaluated for the level of mitosis, the mitotic-specific marker, phospohistamine H3 (PHH3), can be useful for rapid detection of the mitotic level. PHH3 has been used for grading of upper-tract urothelial carcinoma . Since tHG tumors in the WHO 2004 and 2016 classification cover wide ranges of tumors from immediately above low-grade to highly anaplastic tumors. Recently, Cheng and his colleges suggested a four-tiered grading system that included grade 4, which consisted of an anaplastic group, and separating this group from the usual HG , 4, 27. Capillary proliferation has been evaluated in terms of the number of capillary lumina per papillary core that was cross-sectioned, and microvessel density (MVD) has been studied as a prognostic factor in many solid tumors , 29. MVDP-value of 0.2. Most histological parameters played no significant roles in the clinical outcome of PUC-T1, except for divergent histology. This indicated that the presence of divergent differentiation should be considered, particularly in invasive carcinoma. The reason for the reduced significance of divergent histology in the prediction of clinical outcome in PUC-Ta may be related to the low frequency of Ta stage tumors. Aggressive tumors with a divergent histology were more apparent in the invasive stage (T1) and were not usually detected at the Ta stage. Thus, we included divergent histology as one of adverse histological parameters for upgrading. Large cohort studies of PUC-Ta with a significant number of tumors with divergent histologic differentiation may be needed to verify whether this parameter has a clear biological impact.A divergent histology was defined as identifiable histological features differing from the usual urothelial carcinoma. A significant number of high-grade urothelial carcinomas demonstrated glandular or squamous differentiation. In this study, tumors with divergent histology showed a worse clinical outcome than those that were pure urothelial carcinomas. The divergent histology could represent a dedifferentiation with molecular events resulting in a gain of function. Cheng et al. classified tumors with divergent differentiation, such as the nested variant, micropapillary variant, plasmacytoid variant, sarcomatoid carcinoma, small-cell carcinoma, large-cell undifferentiated carcinoma, and pleomorphic giant cell carcinoma, as grade 4 tumors . In our Necrosis or apoptosis may be detected easily in a low-power view, but differentiation between these two features was not easy. In addition, degeneration of the papillary cores with dystrophic calcification could be confused with necrosis.p < 0.05), but that of the latter was less than 0.7. In addition, our proposed grading system was focused on only few, but the most powerful histological parameters, which are not descriptive or subjective, are rather quantifiable and are more reproducible, for practical use. Therefore, our system may be a better option to use as a grading system if it has a similar power for the prediction of the clinical outcome of PUC-Ta.The newly proposed grading system designed here was compared with previous grading systems. Even though the difference in the AUCs between them was not statistically significant, the AUCs of the new grading system were larger than those of the previous grading systems for the prediction of PUC-Ta recurrence. The former AUC was more than 0.7  treatment cannot be separately reviewed among the same grade and stage tumors. However, this study is valuable because it provided a comprehensive analysis of all histological parameters, including mitotic level and count, through a nationwide multicenter study, involving experienced uropathologists.Although diagnostic improvement should be verified by means of a kappa value, we were unable to do so in this study. In the near future, we will collect “gray zone” tumors, with divergent designations by pathologists, and apply the new grading system to determine whether it allows improved diagnosis.The mitotic level based on the initial biopsy appears to be an independent predictor of the PUCa-Ta outcome. This finding could potentially help distinguish between low and high grade tumors in borderline lesions. Therefore, this result may help in selecting patients for a therapeutic strategy, based on the initial biopsy of NMIPUC of the bladder.


ZmLsi1, ZmLsi2 and ZmLsi6, responsible for Si uptake and accumulation. Two weeks after pollination ZmLsi1 and ZmLsi6 genes were expressed, and expression continued until the final developmental stage of the kernels, while ZmLsi2 was not expressed. These results suggest that exclusively ZmLsi1 and ZmLsi6 are responsible for Si transport in various stages of kernel development. Expression level of ZmLsi genes was consistent with Si accumulation within kernel tissues. Silicon was mainly accumulated in pericarp and embryo proper and the lowest Si content was detected in soft endosperm and the scutellum. Correlation linkages between the distribution of Si and some other elements  were found. The relation of Si with Mg was detected in all kernel tissues. The Si linkage with other elements was rather specific and found only in certain kernel tissues of maize. These relations may have effect on nutrient uptake and accumulation.The mineral composition of cells, tissues, and organs is decisive for the functioning of the organisms, and is at the same time an indicator for understanding of physiological processes. We measured the composition of the ionome in the different tissues of maize kernels by element microanalysis, with special emphasis on silicon (Si). We therefore also measured the expression levels of the Si transporter genes  Silene paradoxa under Cu exposure . In their study, various barley varieties grown in the same field were tested, and authors suggested that variation in grain Si concentration is controlled genetically. Genotypic variation in Si concentration of grain may be associated with variation in the ability of root to take up Si, in differences in translocation and accumulation of Si, and in other factors  kernels were obtained from plants cultivated in soil under natural conditions. The plants were grown in sandy-loam soil in the yard of the Department of Plant Physiology, Comenius University in Bratislava and watered regularly from April till October 2015. No insecticides or additional foliar spraying neither addition of nutrients in the form of fertilizers were applied. The concentration of bioavailable Si from the soil (113 ± 15 mg kg-1) was analyzed according to Maize , 1 month after pollination , and mature dry kernel .RNA of the kernel from the 1st developmental stage was extracted from pericarp and the rest of the kernel, because it was possible to extirpate the embryo at this stage. At the 2nd developmental stage, RNA was extracted from pericarp, embryo, and the whole kernel. At the 3rd developmental stage, RNA was extracted from the pericarp and embryo. The Real-time PCR analysis was performed according to our previous work .TM-5 (ZymoReseach). ZmLsi1, ZmLsi2, ZmLsi6, and beta actin genes were amplified by Maxima SYBR Green/ROX qPCR Master Mix (Thermo Fisher Scientific) using Light Cycler II 480 (Roche). The relative changes in gene expression were estimated according to Pfaffl method including amplification efficiency of selected genes . In addition, RNA extraction of the kernel in the 3rd developmental stage was done after 24 h imbibition in sterile conditions. Synthesis of cDNA was performed by ImProm-II Reverse Transcription System (Promega) using Oligo(dT)15 primers and after that, cDNA was purified by DNA Clean & Concentratored genes . After tFigure 1) and the distribution of 11 elements  were observed and analyzed with the Jeol JSM-IT300 scanning electron microscope (SEM) equipped with an energy dispersive X-ray (EDX) analyser. Initial mapping showed relatively low differences in element distribution within kernel tissues , therefore for relative quantification of element distribution all samples were observed at 1000x magnification and 20 kV accelerating voltage, working distance was 11.0 mm. Data were collected for 50 live seconds. As uneven surfaces may heavily distort EDX measurements  were coated with carbon and fixed on aluminum stubs covered with a carbon sticker. Surface conductivity was increased by carbon coating resulting in a uniform thickness of the carbon layer of approximately 60 nm. Kernel tissues . This indicates that data for statistical processing were obtained from 16 individual measurements of each maize kernel zone. Raw data were processed with the TEAM Enhanced, Version 4.3  software and all the values are expressed as weight % of total analyzed elements.Statgraphics Centurion (version 15.2.05) software was used for statistical evaluation. The statistical significance of the means was considered at 0.05 probability level. Analysis of variance (ANOVA) and least significant difference (LSD) were performed on all experimental data sets. Additionally, standard principal component analysis (PCA) and multivariate regression analysis were applied to the study of ionome using Microsoft Excel 2013 and R version 3.0.2 software.F-statistic 420.9 on 43 and 649 degrees of freedom, p-value 0.00) with adjusted R-squared at the level 0.96.The data from EDX measurements were used for the study of ionome. Ionome analysis was first performed by PCA. The original data matrix consisted of 11 element variables  measured in 63 observations differentiated among four different tissue categories . Principal components analysis was performed by singular value decomposition of the centered and scaled data matrix using the function “prcomp” in R version 3.0.2. Variable loadings revealed the correlation structure between original variables and extracted components. Multivariate regression analysis was performed by ordinary least squares estimation procedure “lm” in R version 3.0.2. Structural category embryo proper and Si element were assigned as references, to which all remaining categories and elements, as well as their two-way interaction terms were compared. The resulting model was statistically significant . In this developmental stage, ZmLsi1 and ZmLsi6 genes were expressed; but the ZmLsi2 gene was not. The same pattern of gene expression was observed also in older kernels. In the 2nd stage, only ZmLsi1 and ZmLsi6 were expressed in pericarp, embryo and also in the whole kernel containing endosperm tissue . In the 3rd developmental stage, pericarp tissue was senescent, thus total RNA was not possible to extract. Expression of ZmLsi1 and ZmLsi6 genes was found in the embryo; however, also at this stage ZmLsi2 was not expressed. Analysis of ZmLsi genes corresponds with Si accumulation in adult kernel . EDX analysis showed the highest Si accumulation in embryo proper and pericarp. However, very low Si content was also detected in soft endosperm and scutellum of the embryo (Table 1).Expression of Table 1); while the microelements Zn and Cu showed the lowest content found in the embryo proper. In the scutellum, microelements Zn, Cu, Fe and macroelements Ca and K were significantly higher when compared to the other kernel tissues. Additionally, Ca did not significantly differ between embryo proper, soft endosperm, and pericarp. Soft endosperm tissue was characteristic for very low Mg, P, K, and N content, although some elements  were higher, when compared mainly with the embryo proper. In the pericarp, the concentration of microelements Fe, Cu, and Zn did not differ from values found in embryo proper. Pericarp and soft endosperm contained the highest amount of kernel sulfur. Nitrogen accumulation reached similar values between scutellum and pericarp. Preferentially, N was accumulated in the embryo, while the lowest content was found in soft endosperm.Energy dispersive X-ray measurements showed significantly higher concentration of the macroelements Mg, P, and N in the embryo proper in comparison to all other kernel tissues . Only three elements have significantly different content than Si detected in embryo proper, namely macronutrients K, P, and N. Considering differences between embryo proper and other tissues, content of N and P was significantly below the content of Si in embryo proper in all three remaining categories. Potassium is significantly elevated only in scutellum tissue in comparison to embryo proper . Remaining elements in the scutellum, soft endosperm and pericarp showed statistically indistinguishable contents in comparison to reference element in embryo proper in the presence of N; its content varies at a substantially higher order of magnitude and shadows variation among other elements.Multivariate regression analysis revealed characteristic ionome changes in soft endosperm, scutellum, and pericarp tissues using comparison with Si and embryo proper as a reference category . The values of mineral content were set as source variables and used for PCA. This method revealed three significant principal components (PC); PC1, PC2, and PC3 which explain 69.6% of total variance in maize kernel ionome. The PC1 (SD: 2.13) accounts for 41.2%, PC2 (SD: 1.33) accounts for 16.1% and PC3 (SD: 1.16) accounts for 12.3% of total variance. Projection of eigenvectors in plane defined by PCs reveals complex correlation structure in ionome. Acute angle between eigenvectors of certain elements represents a positive correlation, obtuse angle shows negative correlation and 90° angle refers to mutually uncorrelated elements. Thus, clustering of certain elements displays elemental relationships effectively.In order to introduce additional aspect to ionome analysis, we used standard PCA, capable to determine relationships between specific tissues  and varying element composition in maize kernel . The PC2 is positively correlated only with S, negatively correlated with K, P, Mg, Cl, and Fe . Finally, PC3 is positively correlated with N, Mg, and P and negatively correlated with Cl, S, and Si . In the case of Si clustering with other elements, correlation was found between Si and N, Mg, and P , the other correlation represents Si and Cl together with Cu  and the last cluster consists of Si and S and Cl . Projection of observations in plane defined by PCs suggests the existence of underlying regularity, which is clearly tissue related, as location of four categories mostly remains well-defined in these plots, especially in Figure 5A, which is a plane defined by two most significant PCs. Hence, this demonstrates different accumulation of elements in various kernel tissues. Higher scores of PC1 correspond with observations categorized in soft endosperm and scutellum, lower scores of PC1 correspond with observations categorized in embryo proper and pericarp . Concerning PC2 as a dimension orthogonal with the former one, higher scores of PC2 correspond with observations categorized in soft endosperm and pericarp, lower scores of PC2 correspond with observations categorized in embryo proper and scutellum . Observations are clustered in four quadrants of the scatterplot PC1 vs. PC2. Third dimension is not offering any similar clustering of observations, therefore the interpretation seems to be less tissue related and more hidden in complex multilateral relationships linking together groups of elements by specific functional routes .We have found positive correlation of Cu, Zn, Fe, and K and negative correlation of N, Si, Mg, S, and P with the PC1 , and vice versa. However, the other connected elements, Si and S are detected with above-average content with less certainty in soft endosperm, because the correlation is weak . In scutellum, main correlations consist of Si and Mg, P, and S . In general, tissue of embryo proper contains weaker network of Si-elements relationships in comparison to scutellum, soft endosperm, and pericarp. Here, the most evident relation represents Si and N, the rest of Si relations are much weaker. In pericarp, Si is strongly correlated with P and Mg . The other correlation in pericarp represents weak Si correlation with Cl, Ca, Zn, and Cu.Generalized network representation of ionome in kernel tissues is a novel analytical tool, proposed in order to reveal the structure of multilateral relationships within ionome equilibrium, which can be visualized similar to correlation wheels between pairs of elements. However, unlike standard correlation, pair-wise probability of mutual above average content is not necessarily reciprocal, nor even bidirectional (notice different size of arrow on a link between two elements). Thus, this method reveals varying probability patterns between different tissues corresponding with different functional relationships among elements. For example, in soft endosperm, the most evident reciprocal probability is detected between Si and Cl. It means, that if Si has above average content, there is relative high certainty that also Cl has above average content . Interestingly, Si and P interaction is found only in soft endosperm, scutellum and pericarp tissue and is missing in embryo proper, where it is below threshold. In general, the most abundant relation exists between Si and Mg, P, and Cl. These findings are also supported by PCA, where these relations are clustered with Si, although Si and Cu correlation found in PCA is showed by network representation only in pericarp . Considering all kernel tissues, Si was in relation with macroelements consisting of N, P, S, Ca, and Mg and microelements including Fe, Zn, and Cl.The relation between Si and Mg is found in every kernel tissue, however this correlation is unidirectional only in soft endosperm , while the ZmLsi2 gene was not expressed during kernel development at all. It is known that Lsi1 and Lsi6 transporters (NIP subfamily of aquaporins) are not energetically dependent in comparison to different property of Lsi2 transporter that needs energy to maintain its transport function  that are also tissue specific, except of Si and Mg relation present in all tissues of maize kernel. Considering the fact that many beneficial roles of Si so far are not understood, research about its correlation with other elements in the various tissues becomes especially important. However, additional investigation is necessary to find out the background and role of this correlation in the maize kernel ionome.ZmLsi genes in these tissues. During the developmental stages of kernel, only ZmLsi1 and ZmLsi6 genes were expressed, butZmLsi2 gene not at all. In general, the analysis of the kernel ionome showed that element correlations may be tissue specific and that each investigated tissue  is characteristic by its own ionome with more likely different level of element distribution regulation. The analyses of Si linkage to other elements showed mainly synergic effect (based on probability of above-average content of Si and other element in network representation model) on nutrient accumulation in the kernel tissues. Silicon, as important component of the maize ionome, mainly related with Mg non-specifically in all tissues. The other macroelements P, S, N, K, and Ca related with Si in at least two kernel tissues showing higher degree of tissue specificity. Similarly, Si linkage to microelements Cl, Fe, and Zn was rather tissue specific. These results show that nutrient accumulation in maize kernel may be affected by silicon.We found that high accumulation of Si was detected in the embryo proper and the pericarp what is supported by expression of BB designed the research, performed real-time PCR and wrote the article; SO performed statistical analysis including PCA, multivariate regression and network representation; MV performed EDX analyses and contributed to the design of research and figure preparation; SB considerably contributed to the real-time PCR experiments and analysis; MW and IL enabled EDX analyses; JT and AL supervised the project; and all authors discussed the results and commented on the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Shringasaurus indicus, from the early Middle Triassic of India. The most striking feature of Shringasaurus indicus is the presence of a pair of large supraorbital horns that resemble those of some ceratopsid dinosaurs. The presence of horns in the new species is dimorphic and, as occurs in horned extant bovid mammals, these structures were probably sexually selected and used as weapons in intraspecific combats. The relatively large size and unusual anatomy of Shringasaurus indicus broadens the morphological diversity of Early–Middle Triassic tetrapods and complements the understanding of the evolutionary mechanisms involved in the early archosauromorph diversification.The early evolution of archosauromorphs (bird- and crocodile-line archosaurs and stem-archosaurs) represents an important case of adaptive radiation that occurred in the aftermath of the Permo-Triassic mass extinction. Here we enrich the early archosauromorph record with the description of a moderately large , herbivorous new allokotosaurian,  As part of the outstanding diversification of dinosaurs, multiple theropods and ornithischians  developed elaborate cranial structures, including bony weapons and ornaments7. Cranial weapons have been considered exclusive of dinosaurs during the Mesozoic and generally interpreted as sexually selected traits and evidence of social behaviour7. Here we describe a new herbivorous allokotosaurian stem-archosaur, Shringasaurus indicus gen. et sp. nov., from the early Middle Triassic of central India  in the aftermath of the catastrophic Permo-Triassic mass extinction (~252 Ma) contributed to reshape Mesozoic terrestrial ecosystems and lead to the dominance of dinosaursdia Fig.  that posShringasaurus indicus have been collected from the Denwa Formation of the Satpura Gondwana Basin9  age to the Denwa Formation on the basis of its vertebrate assemblage, but without giving further details. Bandyopadhyay & Sengupta17 proposed an early Anisian age to the upper part of the Denwa Formation based on its brachyopid temnospondyl assemblage and also suggested a late Spathian to earliest Anisian age for the lower part of the unit. Abdala et al.18 also recognized that the upper Denwa Formation can be assigned to the early Anisian based on the presence of a brachyopid temnospondyl related to Hadrokkosaurus bradyi from the lower Anisian Holbrook Member of the Moenkopi Formation, USA. However, those authors also pointed out that the Denwa Formation can be directly correlated with the late Anisian subzone C of the Cynognathus Assemblage Zone of South Africa and the approximately coeval Ashfield Shale of the Wianamatta Group of the Sydney Basin  based on the presence of the temnospondyl genus Paracyclotosaurus. Damiani  argued that the presence of Paracyclotosaurus and the mastodonsaurid Cherninia denwai in the upper Denwa Formation indicate that a younger age (i.e. latest Anisian) is also possible for this unit. Unfortunately, the archosauromorph content of the formation currently does not shed light on the age of the unit. As a result, here we consider the upper Denwa Formation as broadly Anisian in age.The vertebrate fossil assemblage of the Denwa Formation includes the dipnoan Diapsida Osborn, 190319Archosauromorpha Huene, 1946 sensu Dilkeset al., 2015Allokotosauria Nesbitt et al., 2015Azendohsauridae Nesbitt Shringasaurus indicusgen. et sp. nov.‘Śṛṅga’ (Shringa), horn (ancient Sanskrit), and ‘sauros’ (σαῦρος), lizard (ancient Greek), referring to the horned skull; ‘indicus’, Indian (Latin English), refers to the country where such species was discovered.ISIR  780: partial skull roof  with a pair of large supraorbital horns Fig. .Figure 2ISIR 781–1072. Cranial and postcranial bones of at least seven individuals of different ontogenetic stages collected from a single, monospecific 5 metres × 5 metres bone-bed Figs , S2.18, Satpura Gondwana Basin.Near Tekapar village, Hoshangabad district, Madhya Pradesh, India Fig. ; Denwa FRelatively large . The prefrontal and postfrontal are thick and almost exclude the frontal from the border of the orbit. The prefrontal, nasal, frontal, and postfrontal of each side of the skull are fused to each other in large individuals . The skull roof possesses an anteriorly curved, conical bony horn, almost equal in height to the rest of the skull in large individuals .A comprehensive phylogenetic analysis focused on Permo-Triassic stem-archosaurs found ria Fig. . Among a2 and Nesbitt et al.25, with exception of the relationships within Allokotosauria , Pamelaria dolichotrachela was found as the most basal member of Azendohsauridae, and not as the sister-taxon to all other allokotosaurians. The relationships among the other allokotosaurians are congruent with the result of Nesbitt et al.20, including the taxonomic content of the clade Trilophosauridae, in which Teraterpeton hrynewichorum is the sister-taxon of Spinosuchus caseanus and Trilophosaurus buettneri. Within Azendohsauridae, both species of Azendohsaurus are sister-taxa to one another.The topology of the strict consensus tree generated from the MPTs is identical to that recovered by Ezcurraria Fig. . ContrasShringasaurus indicus within Allokotosauria and outside this clade were explored using heuristic tree searches under monophyly or non-monophyly constraints. Two additional steps are necessary to force a sister-taxon relationship between the two Indian allokotosaurians Shringasaurus indicus and Pamelaria dolichotrachela, in which case the clade is sister to all other azendohsaurids. Four extra steps are required to place Shringasaurus indicus as the sister-taxon to all other azendohsaurids, in which case Pamelaria dolichotrachela is found as the sister-taxon to all other allokotosaurians. Alternatively, five extra steps forces Shringasaurus indicus as the most basal azendohsaurid if Pamelaria dolichotrachela is the sister-taxon of Azendohsaurus spp. Fourteen additional steps forces Shringasaurus indicus to be the most basal allokotosaurian and under this constraint Pamelaria dolichotrachela is the sister-taxon of Azendohsauridae + Trilophosauridae. Ten extra steps are necessary to find Shringasaurus indicus as a trilophosaurid, being the sister-taxon to all other members of the clade. Finally, 26 additional steps are required to place Shringasaurus indicus as a non-crocopod archosauromorph (as the sister-taxon of Crocopoda), 25 steps to find it as a rhynchosaur , and 29 steps to find it as a crocopod more derived than rhynchosaurs and allokotosaurians (as the sister-taxon to Boreopricea funerea plus other archosauromorphs).Suboptimal alternative relationships for Prolacertoides jimusarensis and Azendohsaurus laaroussi are pruned a posteriori, the Bremer values are of 4 for both Allokotosauria and Azendohsauridae , but it is two times higher for Azendohsauridae (=4). It is interesting to note that all the absolute and GC bootstrap frequencies within Allokotosauria are higher than 50% Fig. , thus indae Fig. . As a reShringasaurus indicus is its pair of large supraorbital horns  because of the required investment in growth, transport, and maintenance26. Horned individuals of Shringasaurus indicus of different ontogenetic stages show that the size and robustness of the horns were exacerbated towards the adulthood and possess a distinct variability in their orientation and anterior curvature in large individuals  characterize sexually selected –a subset of natural selection in which the resource at stake is mates8– features and, as a result, have been considered as key criteria to recognize secondary sexual characters in the fossil record36. Besides, the presence of substantial variation in the morphology of the horns of Shringasaurus indicus (size and shape) and their potential costliness weakens a species recognition hypothesis as a possible explanation. Species recognition signals have a very low or zero physiological cost 37 and are likely to exhibit minimal variation within a species because high levels of variation would increase the probability of recognition error34. Hence, following the conclusion of previous authors for the elaborate cranial structures of fossil archosaurs 33, a non-adaptive, neutral selection, or species recognition hypothesis are not supported as evolutionary drivers for the origin of the horns of the new species (Supplementary Information).The most striking feature of rns Figs  and 3. Tals Fig. . In extaShringasaurus indicus are found among several amniotes and are mainly used as signals of individual quality and directly as weapons in intraspecific agonistic behaviours , usually in male-male combats to get access to receptive females29. The independent evolution of similar horn shapes and robustness among different groups can be explained as the result of sexual selection acting on the biomechanical performance of weapons29. These lines of evidences, including the similarity with the horns of bovids and chamaeleonids, had been used to infer the origin and function of the horns of extinct dinocephalian synapsids and ceratopsian dinosaurs  that distinctly exceeds the size range of other Early-Middle Triassic archosauromorphs 40, but subsequently successfully exploited by Late Triassic archosauromorphs, such as rhynchosaurs, aetosaurs, and sauropodomorphs42. The large size and unusual anatomy of Shringasaurus indicus broadens the morphological diversity of Early–Middle Triassic tetrapods and complements the understanding of the evolutionary mechanisms involved in their diversification after the Permo-Triassic mass extinction.phs Fig. . Though Shringasaurus indicus were analyzed in the most comprehensive phylogenetic dataset available for Permo-Triassic archosauromorphs2 as modified by Nesbitt et al.25. The matrix was analyzed under equally weighted parsimony using TNT 1.544. A heuristic search with 100 replicates of Wagner trees (with a random addition sequence) followed by TBR branch-swapping (holding 10 trees per replicate) was performed. The best trees obtained from the replicates were subjected to a final round of TBR branch swapping. Zero length branches in any of the recovered MPTs were collapsed. Decay indices  were calculated and a bootstrap resampling analysis, using 1,000 pseudoreplicates, was performed reporting both absolute and GC  frequencies.The relationships of Shringasaurus indicus and three other allokotosaurian species  to the original dataset. Two additional terminals were included after splitting the scorings of “Spinosuchus combined” into Trilophosaurus jacobsi and Spinosuchus caseanus in order to test the synonym hypothesis of Nesbitt et al.20 (Supplementary Information). Two characters were modified, some scorings were changed, and 14 characters were added . The modified data matrix (including “Spinosuchus combined”) includes 88 terminals and 620 characters. The following characters were ordered: 1, 2, 7, 10, 17, 19–21, 28, 29, 36, 40, 42, 50, 54, 66, 71, 75, 76, 122, 127, 146, 153, 156, 157, 171, 176, 177, 187, 202, 221, 227, 263, 266, 279, 283, 324, 327, 331, 337, 345, 351, 352, 354, 361, 365, 370, 377, 379, 398, 410, 424, 430, 435, 446, 448, 454, 458, 460, 463, 472, 478, 482, 483, 489, 490, 504, 510, 516, 529, 537, 546, 552, 556, 557, 567, 569, 571, 574, 581, 582, 588.We added 45 using maximum parsimony in TNT 1.543. Measurements are based on personal observations and published references (Supplementary Information). The median and standard deviation of the femoral length of late Permian to Middle Triassic archosauromorphs  were calculated using the software environment R44.Femoral length, as proxy of body size, was optimized as a continuous characterhttp://zoobank.org/’. The LSIDs for this publication are urn:lsid:zoobank.org:act:DD9F3C0A-1107-4033-8A6E-8B94A4BD9718 and urn:lsid:zoobank.org:act:2CB56E16-EC8B-4691-99BD-A8F3DF4B26E8.This published work and the nomenclatural acts it contains have been registered in ZooBank, the proposed online registration system for the International Code of Zoological Nomenclature. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the Life Science Identifier to the prefix ‘supplemenrary informationdata matrix NEXUSdata matrix TNT


Online interventions for gambling, alcohol, and illegal drug related problems have been developing at a fast pace over the past decade. Yet, little is known about the content and efficacy of interventions provided entirely online for reducing drug/alcohol use and gambling, or about the characteristics of those who use these interventions. This systematic review aims to describe the characteristics of online interventions, their efficacy, and the profile of their clientele. Documentation was mainly obtained through four scientific databases in psychology, technology, and medical research  using three keywords . Of the 4,708 documents initially identified, 18 studies meeting admissibility criteria were retained and analyzed after exclusion of duplicates and non-relevant documents. No study in the review related to problem gambling. The majority of interventions were based upon motivational or cognitive-behavioral theoretical approaches and called upon well-established therapeutic components in the field of addictions. The participants in these studies were generally adults between 30 and 46 years old with a high school education and presenting a high risk or problematic use. More than three quarters of the studies showed a short-term decrease in use that was maintained 6 months later, but only two studies included a 12 months follow-up. Online interventions seem promising and appear to meet the needs of participants who are in the workforce and seeking help for the first time. Long-term efficacy studies should nonetheless be conducted. Diagnostic and Statistical Manual of Mental Disorders , delivered alone or in combination, without necessarily differentiating them  for at risk or problem gamblers or users  and that were assessed for efficacy. This review aims to: (a) identify the objectives of the interventions; (b) describe their characteristics  and modalities ; (c) report the efficacy of interventions in reducing alcohol/drug use and gambling; (d) shed light on participant characteristics; and (e) report the methodological quality of the studies.Studies relating to online treatment of alcohol, drug (excluding prescribed medication) or gambling related problems were identified via the PsycINFO, MedLine, Francis, and INSPEC databases. Two research strategies were used. First, all databanks were searched with three groups of keywords within the entire text:* OR “substance misuse” OR “substance abuse” OR “substance addic*” OR “substance dependence” OR “substance related disorder” OR “drug abuse” OR “drug misuse” OR “drug addic*” OR “drug dependence” OR “alcohol abuse” OR “alcohol misuse” OR “alcohol addic*” OR “alcohol dependence”) AND  AND (treatment* OR intervention* OR therap* OR “online therap*” OR “online intervention*”)For the MedLine database, the group of “Mesh terms” was the following:(“Gambling”[Mesh]) OR “Substance-Related Disorders”[Mesh]) AND  OR “Drug Therapy, Computer-Assisted”[Mesh])http://www.clinicaltrial.org site made it possible to obtain references for unpublished studies and to contact their authors when inclusion criteria were met.A search conducted on the Studies meeting the following criteria were included: (1) written in English or French, (2) published between January 1991 and June 2015 inclusively, (3) involving at least one group whose intervention was entirely online, (4) the intervention targeted the reduction of behaviors or symptoms related to alcohol, drug use, or gambling (5) conducted with users (vs. non-users/abstinents), and (6) used a research design.personalized feedback) or strictly for evaluation, (2) the interventions only targeted relapse prevention or consolidation, and (3) they did not include efficacy data.Studies were excluded if: (1) the online computer support was used for promoting awareness, prevention , No (−) or I don't know (?). Since four criteria were not applicable within the context of this review, only seven of the proposed criteria were retained and rated. The more a study obtains positive ratings, the more it possesses methodological elements of good quality.The methodological quality of the studies was evaluated using a list of 11 criteria recommended by the Cochrane Back Review Group  type of study, (2) methodology, (3) description of participants, (4) intervention objectives, (5) main findings, (7) conclusion, and (8) methodological quality.The study coordinator and five psychology doctoral students participated in data extraction. An undergraduate research assistant counter-verified the information extracted for the 18 studies.To facilitate the reading of the Results section, the 18 studies are referenced using exponents . No study in the review related to problem gambling.Eighteen studies were included in the review and these studies evaluated 22 different interventions in total. Fifteen studies7, 8 are mobile applications for alcohol use, while the others are online interventions .Three of the 22 interventions1, 3, 4, 6, 8, 9, 11, 12, 14, 16, 17), nine upon a motivational approach1, 4, 6, 9, 11, 14, 17, three upon a solution-focused approach15, 18, and three upon a relapse prevention model6, 15, 17. Self-regulation theories3, 4, 12, 17 and planned behavior7 underpin certain interventions, whereas for four interventions2, 5, 10, the theoretical approaches were not indicated.Thirteen interventions were based, at least in part, upon the cognitive and/or behavioral model  targeted decreasing use, seven4, 8, 11, 14, 16 to 18 offered the choice between decreasing use and abstinence, and only one9 targeted abstinence.Of the 22 interventions, 14  used standard self-help therapeutic material: self-report assessment, self-recordings of use, exercises, readings, and videos. This material aimed, for example, to help the individuals identify at risk situations, determine their goals, modify their thoughts, develop problem solving and emotion management strategies, and to prevent relapse. In addition to this material, four interventions1, 9, 11, 18 included online chatting with a clinician, and only one1 involved participating in a discussion forum.The majority of interventions ; the Check your BAC and PartyPlanner App programs. These interventions enabled users to plan and simulate their alcohol use for a given event and receive an estimate of their blood alcohol levels in real-time.Two interventions via mobile applications differed from the other interventions , users' online participation was required only once or twice, whereas the other online interventions lasted between 1 week and 6 months, with suggested utilization of variable frequency .Table 2, 5, 7, military personnel returning from combat4 and workers from a certain workplace10. These studies were excluded from the general description that follows, but are presented in the targeted clientele section.The sample characteristics for each of the studies are shown in Table 1, 8, 11 to 14), however, a greater proportion of men was found in six studies , of which three evaluated an intervention for drug use16 to 18. The mean age of the participants from the 12 studies varied between 30 and 46 years1, 3, 6, 8, 9, 11 to 17.Thirteen studies reported participants' sex. Six of them showed a similar distribution between men and women  indicated occupational status and showed that between 55 and 82% of participants were employed.Six of the 13 studies ; the proportion of participants in a relationship varied between 35 and 61%13.Four studies indicated civil status  reported previous treatment, with percentages varying between 4 and 24% of the sample.Four studies  were applied to individuals with high-risk use or addiction.The majority of the interventions from the 13 studies , however, a majority of men were found in six studies , including the three studies evaluating a drug-related intervention. One study2 indicated that 4.6% of students received prior treatment and another7 showed at risk alcohol use among participants.The participants were young adults, aged on average between 19The participants were men (86%) who had received a prior psychological treatment (61%) and who presented problematic alcohol use.All the participants were employed, the majority were women (78%) and had completed university studies (84%).Table 1 5, 7 to 11, 14 to 18) evaluated post-intervention changes in substance use. These evaluations took place immediately after the intervention or within a delay varying between 2 weeks and 3 months after initial evaluation. The duration of the interventions being variable within a same study and between studies, the evaluation that takes place 3 months after initial evaluation could be considered close to the end of the intervention.Fifteen of the 18 studies  included a 6-month post initial evaluation follow-up. Overall, these studies showed the maintenance of improvements observed shortly after intervention. Only two studies5, 14 evaluated the maintenance of gains 12 months after the initial evaluation, of which one5 showed that the gains were maintained exclusively for women, whereas the other14 study showed maintenance of improvements for all groups, including the group who did not received intervention.Eight of the 18 studies , two satisfied five criteria3, 12, and six met four criteria1, 2, 8, 10, 11. One study met only two criteria13. Half of the 18 studies2, 3, 5, 6, 8, 10, 12, 13, 16 presented insufficient information to determine if they met at least one criterion.Table The goal of this systematic review is to portray psychological interventions that are entirely online for people with alcohol, drug, and gambling related problems.Like the systematic review conducted by Danielsson et al. , this reThe large majority of online interventions for alcohol or drugs were delivered on a web platform, with the exception of three mobile applications. Two of these applications (see Gajecki et al., In regards to the therapeutic objectives targeted by online interventions, the majority of them offer a freedom of choice to the users as to goals of decreasing use or abstinence. They also offer users flexibility when targeting goals for decreased alcohol/drug use. Imposing abstinence may discourage many users from seeking assistance Ursúa,  and favoThe majority of online interventions are based on cognitive-behavioral or motivational approaches; approaches underpinning standard and self-administered psychological treatments for gambling and alcohol/drug related problems (Dutra et al., Contrary to standard cognitive-behavioral treatments that give priority to a clear therapeutic framework (Andersson et al., This review made it possible to extract a profile of participants of online interventions for drug and alcohol use outside of targeted recruitment. Overall, the participants are mostly adults between 30 and 46 years old who are educated and employed. Although less than a quarter of the clientele reported having previously consulted for drug or alcohol related problems, the majority was identified as presenting problematic drug/alcohol use.online format attracts adults between 30 and 46 years old, which indicates that this clientele may be more receptive to technological advances because of their active and regular Internet use (Ducharme, This profile appears similar to that drawn by Postel et al. . The onlThe online interventions generally seem to be efficacious for reducing certain drug and alcohol use behaviors on a short-term basis. The majority of the studies report positive results on at least one substance use indicator following intervention, and some show a medium term maintenance of gains. These results appear to be coherent with other studies evaluating the efficacy of offline self-help treatments (Hodgins et al., This review presents some limitations. One of them relates to the difficulty of the review to meet the final research objective, that of presenting the methodological quality of the studies. Indeed, even though this grid used Cochrane recommended criteria, the evaluation grid used required the withdrawal of nearly a third of the tool's criteria because of the difficulty applying them to the types of studies identified. Another limitation relates to the subjective portrait offered by the review regarding the efficacy of the online interventions since the review's inclusion criteria, as well as the study objectives, did not make it possible to conduct a meta-analysis.The review does however possess strengths, notably the scientific rigor employed during the study selection and database extraction processes. A second strength lays in the selection of interventions included in the review; those available entirely online. By differing from the review of Danielsson et al.  that incIn conclusion, this review shows that, in general, psychological interventions offered completely online for alcohol and drugs do not reinvent the underpinnings of self-administered interventions in regards to both the theoretical approach and their content. The online format represents an alternative way to offer these interventions, which could increase accessibility and attract a clientele who would not consult otherwise. These interventions appear promising and have short-term benefits among their users. However, further research is essential. Firstly, it is primordial to evaluate the efficacy of these interventions while including long-term follow-up measures. Secondly, the interventions offered through mobile applications appear to represent a challenge; they are based on less conventional approaches in regards to addiction and show mitigated results. This type of intervention should be further examined in order to ensure their safety. As such, other rigorous scientific studies are needed to be conducted before integrating them into a treatment program. Finally, development and evaluation of interventions that are entirely online for problem gambling are necessary steps to the diversification of intervention tools for this clientele.Manuscript was written by IG, AG, JM, CJ, and SB. JM and SB helped with the study design, methodological issues and manuscript revision. Data were collected by AG and JM. The study and the manuscript redaction were supervised by IG. All authors made substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work; All authors worked on drafting the work or revising it critically for important intellectual content; and all authors approved the version to be published and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Supplemental Digital Content is available in the text In the double-blind, randomized, controlled trial, we aimed to evaluate the effects of compound tufuling oral liquid (CoTOL) on serum uric acid (sUA) levels and recurrence of acute gouty arthritis in intercritical and chronic gout treatment.A total of 210 patients with gout were screened from 8 hospitals to observe the sUA and acute gouty arthritis recurrence rate-reducing effects of CoTOL in intercritical and chronic gout during a 12-week treatment. We treated 139 and 71 patients with CoTOL and the placebo, respectively, and evaluated their sUA levels, acute gouty arthritis recurrence rate, and adverse events at week 0, 6, and 12.P = 0.004). The average decrease rate of sUA was 12.76% (95% CI: 9.82%–15.70%) and 4.57% (95% CI: 0.42%–8.71%) in the treatment and control groups, respectively (P = 0.004), and the gouty arthritis recurrence rate of the treatment group was lower than that of the control group . Severe adverse events were not observed in either groups, and fewer leucopenia incidences were observed in the treatment group than those in the control group .Twenty-five and 12 patients in the treatment and control groups, respectively, had interrupted treatments, whereas 114 and 59 cases, respectively, completed their treatments. At the end of the 12-week treatment, the average decrease in sUA was 74.26  and 28.81 μmol/L (95% CI: 4.91–52.71 μmol/L) in the treatment and control groups, respectively (CoTOL reduced sUA levels and effectively prevented acute arthritis recurrence in intercritical and chronic gout without serious adverse events. In affected individuals, urate crystals are deposited in the joints, leading to acute gouty arthritis. The prevalence of gout is increasing worldwide and was 1.4% in the UK and Germany from 2000 to 2005, and increased from 6.7 per 1000 inhabitants in 2005 to 9.1 per 1000 inhabitants in 2009 in Italy. A similar trend of increase in gout prevalence has also been observed in China. In Taiwan, the prevalence of a history of gout was found to be 15.2% (25/165) and 4.8% (11/231) in the Aboriginal men and women, respectively, compared with a prevalence rate of 0.3% in non-Aborigines. In 2002, the age-standardized prevalence was 25.3% for hyperuricemia and 0.36% for gout in adults aged 20 to 74 years in Qindao, China.Gout is a metabolic rheumatism characterized by hyperuricemia and recurrent acute gouty arthritis, which is difficult to cure and relapses easily. The etiopathogenesis of gout is related to genetic whereas hypouricemic agents  are used for chronic gout. Unfortunately, although these drugs show some therapeutic effects, they also cause numerous side effects. For example, colchicine, which inhibits leukocyte migration and lactic acid production to decrease inflammatory responses and urate crystal deposition, may also induce abdominal pain, nausea, vomiting, diarrhea, bone marrow depression with aplasticanemia, agranulocytosis, thrombocytopenia, peripheralneuritis, alopecia, reversible azoospermia, myoneuropathy and myopathy, and rhabdomyolysis. NSAIDs may exacerbate epeptic ulcers, renal failure, hypertension, and cardiovascular disease. Similarly, glucocorticoids may exacerbate peptic ulcers, hypertension, hyperlipidemia, and diabetes, which results in patients with gout discontinuing their treatment. Allopurinol is also limited by adverse events such as hypersensitivity reactions and can even cause death. Therefore, it is of great concern to find effective herb medicines and nonpharmacological treatment methods.Conventional western medicines used to treat acute gout include anti-inflammatory and analgesic agents ,,25 However, the absence of large sample, double-blind, randomized controlled trials (RCTs) affects the reliability and replicability of their conclusions. Therefore, designing and conducting a large-sample double-blind RCT to demonstrate the effectiveness of TCM for gout treatment is of great importance.In China, numerous traditional Chinese medicines (TCMs) have been used to treat gout for thousands of years. Several studies comparing Chinese herbal decoctions and traditional Western medicines for gout treatment have been reported over the past decade.–30 In the present study, we sought to further explore the effect of CoTOL on the sUA levels of patients in the intercritical and chronic gout phase, and the recurrence rate of acute gouty arthritis by conducting a double-blind, multicenter, placebo-controlled, RCT (S1 File).Our previous studies showed that CoTOL , which we developed after years of clinical and experimental research, reduces serum uric acid (sUA) levels in intercritical and chronic gout and alleviates acute arthritis without severe adverse reactions even after long-term use.22.1 Informed consent was given to every participant, and all participants had provided the written informed consent.The trial was designed as a prospective, multicenter, randomized, placebo-controlled, and double-blind trial of CoTOL Table  in patie2.2 based on the patient's medical history, without gouty arthritis attack at the time of enrolment, and willing to participate in the trial and sign informed consent certificates. The exclusion criteria included patients: with acute gouty arthritis at the baseline, administered a uricosuric medicine within 2 weeks, with secondary gout, with serum creatinine (Scr) >133 μmol/L or urinary calculi, with serious organ dysfunction, mental illness, or cancer, and body mass index >50 kg/m2 or alcoholism. The suspension criteria included: occurrence of serious adverse drug reactions (ADRs) leading to the study team deciding to suspend the patient from the study after adequate evaluation; patients whose sickness worsened after treatment or whose syndrome was influenced by unexpected factors and should be treated as invalid cases; treatment with other gout therapies during the study period, which rendered the participant as an invalid case; and an accumulated treatment interruption of 28 or 21 days at a stretch was regarded as an invalid case. The withdrawal criteria included patients who did not complete the whole treatment course because of serious ADRs or other possible reasons. The cases that were considered withdrawals were managed as follows:(1)After patients had withdrawn from the study, the researcher coordinators contacted them using every possible method such as personal visits, telephone, or mail to identify their reasons for withdrawing. In addition, the time of their last medication was recorded, and all the possible evaluations were completed.(2)Patients who withdrew from the study because of allergies or other ADRs were properly treated with other therapies.(3)All the test data of the withdrawn cases were properly kept as records for use in the total analysis and calculation.The inclusion criteria included: male patients, aged 18 to 60 years, with hyperuricemia (blood uric acid [UA] >480 μmol/L), meeting the American College of Rheumatology Classification standard for primary gout revised in 1977 The participants were recruited from June 9, 2012 to May 31, 2013 in 9 hospitals, which were the Rheumatism Institute, Zhejiang Chinese Medical University, Hangzhou, Zhejiang, China; Department of Rheumatism, the Second Affiliated Hospital of Zhejiang University Medical College, Hangzhou, Zhejiang, China; Department of Rheumatism, Zhejiang Hospital, Hangzhou, Zhejiang, China; Department of Rheumatism, Chinese Medical Academy Gate Hospital, Beijing, Beijing, China; Department of Rheumatism, Zhejiang People's Hospital, Hangzhou, Zhejiang, China; Department of Rheumatism, Sir Run Run Shaw Hospital of Zhejiang University Medical College, Hangzhou, Zhejiang, China; Department of Rheumatism, the First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, Zhejiang, China; Department of Public Healthy, Medical College, Zhejiang University, Hangzhou, Zhejiang, China; and Department of Rheumatism, the Second Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, Zhejiang, China.http://www.chictr.org/ChiCTR-TRC-12002245 website. The authors confirm that all ongoing and related trials for this drug/intervention are registered.This trial was registered at the Chinese Clinical Trial Registry on June 9, 2012 before participant recruitment and is available on the 33.1 A moderate treatment duration of 12 weeks was chosen out of consideration for patients receiving placebo and for ethical reasons.The sample size of the measurement data was calculated by using the superiority test: 3.2This study adopted the envelope random method. With each center as a block, the Quality Control Department of the Affiliated Hospitals of Nanjing University of Chinese Medicine used the Statistical Analysis Software (SAS) version 9.2  to generate randomized numbers for each group and patient to determine whether they should be assigned to the treatment or control group.3.3The double-blind method was used to mask the treatments. The researcher coordinators adjusted the basic recipe based on each patient's temporary symptoms and subsequently informed the specially assigned drug management personnel to adjust the procedures afterward. The drug management personnel first obtain each patient's random and group numbers and then informed the pharmacist to decoct their specific treatment recipe or placebo based on the patient's group information , respectively. The age, course of gout, frequency of acute arthritis over 1 year after treatment, joint function, and comorbidities were recorded for each patient.The treatment recipe was decocted twice by refluxing with water  for 1 hour, and then it was vacuum packed into two 250-mL packages. A rapid-performance liquid chromatography identification method was used to authenticate the compound recipe. The control group patients were treated with a placebo consisting of 20 g each of charred millet sprout  and fructus hordei germinatus , 12 g of charred fructus crataegi , and 3 g of edible bitter principle, which each have no therapeutic effects on gout. The placebo solution was prepared using the same procedure used for the study drug and vacuum packed into two 250-mL packages, and the color, taste, smell, and physical form of the placebo were the same as those of the treatment recipe.55.1The primary outcomes were the average decrease and decrease rate of sUA at the end of the 12-week treatment. The average decrease was calculated by using the following formula: C = a − b . The decreased rate was calculated by using the formula: R(%) = 100 × (a − b)/a . The secondary outcome was the decrease in the frequency of recurrent joint swelling or pain. During the 12-week study, fasting venous blood samples were collected in the morning to monitor the biochemical indexes (including sUA). A complete blood count measurement was performed at the baseline as well as at the end of 6 and 12 weeks. The frequency of recurrent joint swelling and pain was recorded at the 6- and 12-week treatment points.5.2The adverse events (AEs) including drug-induced liver or renal injury  and hematological parameters , total cholesterol (TC), high-density lipoprotein cholesterol [HDL-c], and triglyceride [TG]), and vital signs (systolic blood pressure and diastolic blood pressure) were recorded at all scheduled clinic visits.5.3http://www.njecdm.com) administered by the Quality Control Department of the China State Clinical Trial Center of TCM, which was assigned by the State Administration of TCM of the People's Republic of China. The data management was performed by using the web-based clinical trial management platform, ResMan. The data were blindly managed and analyzed by the clinical and statistical staff of the Department of Public Health, Medical College, Zhejiang University.Each patient's clinical information was recorded in a case report form (CRF). Then, 2 data entry staff members entered the data into the electronic CRF on the e-clinical study website  version 18.0 for Windows  and the SAS software version 9.2.The dataset was first exported from the electronic CRF on the e-clinical study website. The intention-to-treat (ITT) or safety set (SS) analysis included patients who responded to at least 1 follow-up survey, and the per-protocol population set (PPS) analysis included patients who adhered to the treatment protocol. The values are presented as the median (interquartile range) for continuous variable and proportion or ratio for categorical variables. The differences in the decreased value or decreased rate of the sUA between the groups were analyzed using the Wilcoxon signed-rank test. The 66.1The flow of the participants through the trial is shown in Fig. 6.2Table 6.3z = −2.87, P = 0.004, Wilcoxon rank sum test). Moreover, the average decreased rate of the sUA was 12.76% (95% CI: 9.82%–15.70%) in the treatment group versus 4.57% (95% CI: 0.42%–8.71%) in the control group  in treatment group versus 28.81 μmol/L (95% CI: 4.91–52.71 μmol/L) in the control group . Furthermore, the average decrease rate of sUA was 12.96% (95% CI: 10.01%–15.90%) in the treatment group versus 4.57% (95% CI: 0.42%–8.71%) in the control group  in treatment group versus 28.81 μmol/L (95% CI: 4.91–52.71 μmol/L) in the control group (χ2 test) revealed that the recurrence rate of gouty arthritis (joint swelling or pain) in the treatment group was lower than that in the control group. The specific values were: from baseline to the end of week 6, 32.14% and 48.28% in the treatment and control groups, respectively, P = 0.04; from the end of week 6 to the end of week 12, 22.02% and 50.88% in the treatment and control groups, respectively, P < 0.001; and from baseline to the end of week 12, 38.53% and 63.16% in the treatment and control groups, respectively, P = 0.003, Fig. χ2 test) revealed that the recurrence rate of gouty arthritis in the treatment group was lower than that in the control group. The specific values were: from baseline to the end of week 6, 30.77% and 44.44% in the treatment and control groups, respectively, P = 0.062; from the end of 6 to the end of week 12, 21.93% in the treatment and control groups, respectively; P = 0.000; and from baseline to the end of week 12, 39.50% and 63.16% in the treatment and control groups, respectively, P = 0.003, Fig. The PPS analysis , Scr, TC, TG, HDL-c, and occult blood stool were measured. Except for the leukocyte count, no obvious abnormalities or indications of worsening symptoms were observed in the 2 groups during the treatment. Fewer cases of leukopenia were observed in the treatment group than in the control group , acute gout, and chronic gout (including the development of chronic gouty arthritis and gouty stones) phases. The prevalence of gout is increasing worldwide –30 we adopted a double-blind, placebo-controlled, multicenter, randomized trial design to determine the effect of CoTOL. Our results indicate that the decrease in the sUA level of the treatment group was significantly higher than that of the control group. Furthermore, the frequency of recurrent joint swelling or pain in the treatment group was obviously lower than that in the control group, whereas the number of patients with an exacerbated white blood cell count was lower in the treatment group than it was in the control group. In addition, severe AEs did not occur in either group. Two and 8 patients in the treatment and control groups, respectively, experienced mild AEs. Therefore, our results indicate that CoTOL reduced the sUA levels of patients in the intercritical and chronic gout phases and prevented the recurrence of acute gouty arthritis with a better efficiency than the placebo did.Based on previous studies,Smilax glabra, Tufuling), Rhizoma dioscoreae collettii , Curcuma longa , Herba siegesbeckiae , and Rhizoma corydalis , Semen coicis , Loranthus parasiticus , and Stigma maydis . Several pharmacological studies have shown that these herbs may have positive effects on gout. For example, Rhizomasmilacis glabrae has anti-inflammatory and analgesic actions. As a flavonoid compound isolated from Rhizoma smilacis Glabrae, astilbin has anti-inflammatory effects. In addition, astilbinis effective in preventing hyperuricemia and nephropathy by increasing the urinary UA level and fractional excretion of urate, preventing renal damage by the expression of transforming growth factor-β1 and connective tissue growth factor, and inhibiting the formation of monosodium urate as well as the production of prostaglandin E2 and interleukin-1. These findings provide strong evidence supporting the use of astilbin as a safe and promising lead compound for the prevention of hyperuricemia and gouty nephropathy. D tokoro Makino and its saponin ingredients, which have been shown to decrease foot swelling in mice and rats and heighten the pain threshold of mice, have positive effects on gouty arthritis induced by sodium urate by reducing sUA.,40 Herba siegesbeckiae has anti-inflammatory and analgesic effects.–43 Rhizoma corydalis has analgesic effects and is effective for treating gastric ulcer.,45C longa, which contains 2 classes of secondary metabolites namely curcuminoids and essential oils, possesses effective inhibitory activity against xanthine oxidase as well as anti-inflammatory and antiarthritic effects.–49 Because of its strong antioxidant capacity and recovery of cisplatin-induced nephrotoxicity, L parasiticus exhibits potent anti-inflammatory and antinephrotoxic activities,–52 which may be beneficial for reducing the recurrence of gouty arthritis and preventing urate-induced renal damage. Semen coicis shows analgesic and anti-inflammatory properties.,54 Stigma maydis (corn silk), which contain saponin, alkaloid, flavones, volatile oil, mucilage, Vitamins B, C, and K, Silicon, increases the urinary output and the percentage the passage of urinary stones through the urinary tracts without decomposed stones, lower the total amount of acid in the urine,,56 and inhibit xanthine oxidase, Therefore, Stigma maydis play an active role in treating gout by inhibiting the production of sUA, promoting the excretion of UA, and preventing the formation of kidney stones.CoTOL consists mainly of Rhizoma smilacis Glabrae (Therefore, we hypothesized that CoTOL might lower sUA and reduce the recurrence of gouty arthritis. The present study clearly demonstrates that the median sUA levels of patients treated with CoTOL were significantly decreased compared to those of the patients treated with placebo after 12 weeks, whereas the recurrence rate of joint swelling or pain in the treatment group was lower than that in the placebo control group. Moreover, severe AEs did not occur in either group. Only 2 participants experienced mild AEs and three manifested leukopenia in the treatment group, which was unrelated to CoTOL.8In conclusion, our findings suggest that CoTOL has significant therapeutic potential not only by reducing the sUA level but also by decreasing the frequency of recurrent joint swelling or pain without obvious AEs. The study has limitations including the fact that the observation period was limited to 12 weeks, and no positive control was used. Future studies should involve a trial with a longer treatment course and a positive control drug to provide further evidence of the efficacy of CoTOL in treating gout. Furthermore, its mechanisms of action also need to be investigated.


An eruption cyst (EC) is a benign, developmental cyst associated with a primary or permanent tooth. This paper presents 66 ECs in 53 patients who reported to 3 different centers in Turkey between 2014-2015.53 patients  with 66 ECs were diagnosed and treated over a 1-year period. The mean age of patients was 5.4 years . Clinical examination and periapical radiographs were used to establish diagnosis. Age, gender, site, history of trauma and type of treatment were recorded.Of the 66 ECs diagnosed in 53 patients, more than half (56.6%) were located in the maxilla, with the maxillary first primary molars the teeth most commonly associated with ECs (30.3%). Multiple ECs were diagnosed in 13 of the 53 patients. ECs had previously diagnosed in the primary dentition of 2 patients, 3 patients reported a history of trauma to primary teeth. In the majority of patients , no treatment was provided, whereas surgical treatment was provided in the remaining 7 cases (13.2%).Eruption cysts are usually asymptomatic and do not require treatment;. however, if the cyst is symptomatic, it should be treated with simple surgical excision.Key words:Odontogenic cyst, children, eruption cyst, oral pathology. An eruption cyst (EC) is a benign, developmental odontogenic cyst that accompanies an erupting primary or permanent tooth, forming shortly before the tooth’s appearance in the oral cavity . In the Clinically, an EC appears as a soft, translucent, dome-shaped lesion filled with blood or a clear fluid overlying the crown of an erupting tooth . ECs norThere are numerous studies in the dental literature on ECs ,5,6,8-12This study was conducted with 53 patients ranging in age from 5 months to 11 years old who were referred to 1 of 3 different dental centers in Turkey for the management of an EC. The study was approved by the human ethics committee of Ondokuz Mayıs University (No:2016/212-14), and all procedures were conducted in accordance with the 1975 Helsinki Declaration (as revised in 2008). Diagnosis was established based on clinical examination and, if the child showed adequate cooperation, periapical radiographs. Patients with a superficial swelling located over the crown of a tooth in the eruption stage shortly before the emergence of the tooth into the oral cavity were included in the study if radiographic characteristics showed no bone involvement. Swellings varied in size and color from transparent to blue, purple and blue-black  were calculated and recorded.In total, 53 patients with 66 ECs were included in the study. Mean age of patients was 5.4 years (age range: 5 months-11 years), and the majority of patients 31 (58.4%) were male . ECs were associated with permanent dentition in 58.5 % of the cases, the primary dentition in 41.5 %. Clinical characteristics of the cases associated with permanent and primary dentition are given in Clinically, most ECs presented as a raised, bluish gingival mass on the alveolar ridge. EC size varied depending upon whether the cyst was associated with a primary or permanent tooth.The majority  of ECs were not associated with any discomfort and were thus initially followed-up without active intervention with the exception of recommending massaging the lesion during the observation period. The teeth associated with all these lesions erupted without any complications in approximately 2 months. However, in 7 cases (13.2%), the presence of the cyst caused a dull, aching pain on mastication, and both patients and their parents were concerned about the unaesthetic appearance. Therefore, in these cases, the ECs were treated by incising them and draining their contents. Of these 7 cases, surgical exposure was carried out under local anesthesia in 3, whereas 4 cases required sedation with nitric oxide due to the age and uncooperative nature of the patient. In all 7 cases, teeth were clinically apparent within a few weeks following surgical intervention.While the process of dental eruption tends to progress without any major events, it can be a stressful experience, especially if something unusual occurs during this period. The sight of an EC, a type of lesion associated with erupting teeth, can cause panic in parents, who may jump to the conclusion that this often large, bluish-purple or bluish-black lesion is some kind of malignant tumor . IncreasThe etiology of ECs remains unknown. While some authors believe degenerative cystic changes in the enamel epithelium or remaining dental lamina occurring during the process of amelogenesis play a role in the development of ECs ,8, otheret al, who reported multiple ECs in a 4-year-old boy with Menkes Kinky Hair Disease who was treated with an anticonvulsant (diphenylhydantoin)  . In the The gender predilection of EC is controversial. Some authors have reported higher prevalence among females , whereasEC usually presents in the first or second decades of life , with thPrevious studies suggested that the majority of ECs are located in the incisal and molar areas, followed by the canine and premolar areas ,16. In oEC is not detectable on radiographic examination because it is difficult to distinguish the cystic space of eruption cyst because both the cyst and tooth are directly in the soft tissue of the alveolar crest. Although dentigerous cysts (DCs) were considered in the differential diagnosis, DCs include a well-defined, unilocular radiolucent area, whereas no bone involvement is seen with ECs ,7,10,17.In most cases, EC subsides spontaneously; therefore, the recommended treatment is close monitoring with follow-up throughout the eruptive process of the involved teeth ,11,12. W