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Canonical Wnt signaling regulation is essential for controlling stemness and differentiation of mesenchymal stem cells (MSCs). However, the mechanism through which canonical Wnt-dependent MSC lineage commitment leads to chondrogenesis is controversial. Some studies hypothesize that inhibition of canonical Wnt signaling induces MSC chondrogenic differentiation, while others support that the pathway should be activated to achieve MSC chondrogenesis. The purpose of the present review is to analyze data from recent studies to elucidate parameters regarding the role of canonical Wnt signaling in MSC chondrogenic differentiation.

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A complete package of functions in the R-language has been written for professional separation optimization of complex mixtures of ionized and/or non-ionized solutes. The package includes functions for (a) base-line correction of experimentally recorded chromatograms, (b) modeling of chromatographic peak shapes and retention data, (c) prediction of the retention time of the test analytes and/or their chromatograms, and (d) separation optimization under either isocratic or single and/or double gradient elution conditions by changing the organic modifier(s) content and/or eluent pH. The optimization functions presented in this study offer two different modes for selection of optimal separation conditions: automatic and manual mode. In the automatic mode, the optimal separation conditions are determined by maximizing the resolution within separation time preset by the analyst. In the manual mode, the optimal separation conditions are selected via scatter or contour plots. The foreknowledge of the precise dependence of resolution and separation time upon one or two retention parameters of interest, provided by the proposed computer-assisted separation optimization method, gives chromatographers a feel of confidence for the selection of the optimal conditions for a desired separation. An illustrative video given in the Supplementary material may encourage a novice practitioner in R (software) programming language to follow the proposed separation optimization procedure in a real HPLC analysis.

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INTRODUCTION: Microfracture does not lead to complete healing of full-thickness cartilage defects. The aim of this study was to evaluate the effect of modifying Wnt/ß-catenin signaling following microfracture, on the restoration of a full-thickness cartilage defect in a rabbit model. The modification of the canonical Wnt pathway was achieved through per os administration of lithium carbonate, which is an intracellular inhibitor of glycogen synthase kinase 3-ß (Gsk3-ß) and therefore induces Wnt/ß-catenin signaling. MATERIALS AND METHODS: Full-thickness cartilage defects of 4 mm in diameter were created in the patellar groove of the right femurs of 18 male New Zealand white rabbits. The rabbits were divided into three groups of six (n = 6) based on post-surgery treatment differences, as follows: microfracture only (group 1), microfracture plus lithium carbonate 7 mM in the drinking water for 1 week (group 2), microfracture plus lithium carbonate 7 mM in the drinking water for 4 weeks (group 3). All animals were sacrificed 9 weeks after surgery. The outcome was assessed histologically, by using the International Cartilage Repair Society (ICRS) visual histological scale. Immunohistochemistry for type II collagen was also conducted. RESULTS: Statistical analysis of the histological ICRS scores showed that group 3 was significantly superior to group 1 in four out of six ICRS categories, while group 2 was superior to 1 in only two out of six. CONCLUSION: The combination of microfracture and systematic administration of lithium carbonate 7 mM for 4 weeks shows statistically significant superiority in four out of six ICRS categories compared with microfracture only for the treatment of full-thickness cartilage defects in a rabbit experimental model.

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BACKGROUND: Most acromioclavicular (AC) joint injuries occur in men in their third decade of life during high-speed or high-impact body contact sports. The management of acute complete AC joint dislocation is surgical. Current surgical techniques include anatomic reconstruction of the main restraints of the AC joint and aim to improve functional outcomes and to reduce the complication rate. METHODS: We present 10 cases of acute type V AC joint dislocation in professional athletes treated surgically with anatomic reconstruction of the coracoclavicular and AC ligaments and augmentation with the use of a synthetic polyester tape. The minimum follow-up of the patients was 2 years (mean, 48 months; range, 24-86 months). The postoperative functional outcome was assessed at 1 year and 2 years using the Constant-Murley, American Shoulder and Elbow Surgeons, and modified University of California-Los Angeles scoring systems. RESULTS: In all cases, the postoperative scores were significantly improved (P < .005 in all comparisons with the preoperative scores), and all patients returned to their preinjury high level of activity 6 months postoperatively. Radiographs at 1 month and 6 months revealed the maintenance of reduction. There were no complications. CONCLUSION: According to the results of our series of patients, demanding cases of acute AC joint dislocation Rockwood type V, in professional athletes, require anatomic fixation of both coracoclavicular and AC ligaments for return to sports as soon as possible and at the preinjury level of performance.

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Type 2 diabetes mellitus affects nearly four hundred million people worldwide, and one of its major complications is cardiovascular disease. The evaluation of the effectiveness and safety of antidiabetic medication has been a challenging issue. Large clinical trials of new antidiabetic medications have used the non-inferiority approach to ensure primary safety of the drug before its incorporation into clinical practice. Currently, the trend is to prove superiority, that is, to prove that the new drug has additional beneficial effects to those of standard medications. In this review, we present the results of recent clinical trials on type 2 diabetes mellitus medications and outline what can be anticipated from ongoing clinical trials.

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A package of Excel VBA macros have been developed for modeling multilinear gradient retention data obtained in single or double gradient elution mode by changing organic modifier(s) content and/or eluent pH. For this purpose, ten chromatographic models were used and four methods were adopted for their application. The methods were based on (a) the analytical expression of the retention time, provided that this expression is available, (b) the retention times estimated using the Nikitas-Pappa approach, (c) the stepwise approximation, and (d) a simple numerical approximation involving the trapezoid rule for integration of the fundamental equation for gradient elution. For all these methods, Excel VBA macros have been written and implemented using two different platforms; the fitting and the optimization platform. The fitting platform calculates not only the adjustable parameters of the chromatographic models, but also the significance of these parameters and furthermore predicts the analyte elution times. The optimization platform determines the gradient conditions that lead to the optimum separation of a mixture of analytes by using the Solver evolutionary mode, provided that proper constraints are set in order to obtain the optimum gradient profile in the minimum gradient time. The performance of the two platforms was tested using experimental and artificial data. It was found that using the proposed spreadsheets, fitting, prediction, and optimization can be performed easily and effectively under all conditions. Overall, the best performance is exhibited by the analytical and Nikitas-Pappa's methods, although the former cannot be used under all circumstances.

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In the present work the retention of three highly polar and ionizable solutes - uric acid, nicotinic acid and ascorbic acid - was investigated on a mixed-mode reversed-phase and weak anion-exchange (RP/WAX) stationary phase in buffered aqueous acetonitrile (ACN) mobile phases. A U-shaped retention behavior was observed for all solutes with respect to the eluent organic modifier content studied in a range of 5-95% (v/v). This retention behavior clearly demonstrates the presence of a HILIC-type retention mechanism at ACN-rich hydro-organic eluents and an RP-like retention at aqueous-rich hydro-organic eluents. Hence, this column should be promising for application under both RP and HILIC gradient elution modes. For this reason, a series of programmed elution runs were carried out with increasing (RP) and decreasing (HILIC) organic solvent concentration in the mobile phase. This dual gradient process was successfully modeled by two retention models exhibiting a quadratic or a cubic dependence of the logarithm of the solute retention factor (lnk) upon the organic modifier volume fraction (φ). It was found that both models produced by gradient retention data allow the prediction of solute retention times for both types of programmed elution on the mixed-mode column. Four, in the case of the quadratic model, or five, in the case of the cubic model, initial HILIC- and RP-type gradient runs gave satisfactory retention predictions of any similar kind elution program, even with different flow rate, with an overall error of only 2.5 or 1.7%, respectively.

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In the present study a series of 45 metabolite standards belonging to four chemically similar metabolite classes (sugars, amino acids, nucleosides and nucleobases, and amines) was subjected to LC analysis on three HILIC columns under 21 different gradient conditions with the aim to explore whether the retention properties of these analytes are determined from the chemical group they belong. Two multivariate techniques, principal component analysis (PCA) and discriminant analysis (DA), were used for statistical evaluation of the chromatographic data and extraction similarities between chemically related compounds. The total variance explained by the first two principal components of PCA was found to be about 98%, whereas both statistical analyses indicated that all analytes are successfully grouped in four clusters of chemical structure based on the retention obtained in four or at least three chromatographic runs, which, however should be performed on two different HILIC columns. Moreover, leave-one-out cross-validation of the above retention data set showed that the chemical group in which an analyte belongs can be 95.6% correctly predicted when the analyte is subjected to LC analysis under the same four or three experimental conditions as the all set of analytes was run beforehand. That, in turn, may assist with disambiguation of analyte identification in complex biological extracts.

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Expressions for the retention time of ionogenic analytes eluted under multilinear double pH/solvent-gradients in reversed-phase liquid chromatography are developed by dividing each gradient profile into a finite number of subportions, where the solute retention factors or their logarithms vary linearly with time. To test the theory, two series of experimental gradient retention data of amino acid OPA derivatives were analyzed: The first one was a monolinear or bilinear pH-gradient data set obtained in eluents with different but constant organic modifier contents, whereas the second data set comprised retention data of combined pH/organic solvent-gradients, where the organic content was changed linearly with time but the variation of pH exhibited a curved form approximated by five linear subportions. It was found that the derived expressions describe these experimental retention data with high accuracy, since under double pH/solvent-gradients the overall errors in the fitted and predicted retention times were 1.9% and 1.7%, respectively, whereas under simple pH-gradients these errors were 0.9% and 2%, respectively.

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This work provides a theoretical basis for an empirical model proposed in Fasoula et al. (2013) [2] for the retention of monoprotic acids/bases during linear organic modifier gradient runs performed with a fixed change of organic content but for different gradient durations and in different eluent pHs. Based on the analytical expressions derived in the above publication for the retention of monoprotic solutes under organic modifier gradients in different mobile phase pHs it was proved that the dependences of retention time of all monoprotic acids/bases upon each of two factors governed the gradient elution under consideration are always the following: practically linear upon gradient duration (for constant pH) and sigmoid upon pH (for constant gradient duration).

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The influence of eluent pH on retention of ionizable analytes during organic modifier gradients in reversed-phase high performance liquid chromatography is studied. Two approaches are examined for the retention prediction of solutes under organic modifier gradient conditions at any constant mobile phase pH: In the first approach an analytical solution of the fundamental equation of gradient elution in linear organic modifier gradients for monoprotic acids/bases under certain assumptions is proposed. The second approach is based on an empirical model arising from the evaluation of the gradient retention data. Both approaches were successfully applied to describe the retention behavior of 16 OPA derivatives of amino acids obtained in 19 simple mono-linear organic modifier gradient runs performed between two given acetonitrile contents with different gradient duration and at different eluent pHs, in a particular pH range where amino acids behave as weak monoprotic acids. Further, this study provides a reliable way for optimizing gradient separations of ionogenic compounds with respect to mobile phase pH.

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An approach for retention modeling of double pH/organic solvent gradient data easily generated by automatically mixing two mobile phases with different pH and organic content according to a linear pump program is proposed. This approach is based on retention models arising from the evaluation of the retention data of a set of 17 OPA derivatives of amino acids obtained in 27 combined pH/organic solvent gradient runs performed between fixed initial pH/organic modifier values but different final ones and for different gradient duration. The derived general model is a ninth parameter equation easily manageable through a linear least-squares fitting but it requires eighteen initial pH/organic modifier gradient experiments for a satisfactory retention prediction in various double gradients of the same kind with those used in the fitting procedure. Two simplified versions of the general model, which were parameterized based on six only initial pH/organic modifier gradients, were also proposed, when one of the final double gradient conditions, pH or organic content was kept constant. The full and the simplified models allowed us to predict the experimental retention data in simultaneous pH/organic solvent double gradient mode very satisfactorily without the solution of the fundamental equation of gradient elution.

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Three retention models for liquid chromatography are developed using principal component analysis (PCA). It is shown that they exhibit features similar to that of the model based on linear solvation energy relationship (LSER). However, the fitting performance of the PCA models is better than that of the LSER model, the performance of which can be considerably improved by the use of artificial neural networks. In addition, the possibility of using the proposed models as well as the LSER model to predict the retention times of solutes under chromatographic conditions at which these solutes have never been studied is also examined by means of three data sets of analytes consisting of non-polar compounds to polar compounds with a variety of functional groups.

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The work carried out on the linear pH-gradient is critically reviewed in combination with the development of new expressions for the retention time of ionizable analytes. It is shown that under ideal linear pH-gradient conditions the better a model describes the 1/k versus pH plot, where k is the retention factor of the analyte, the better performance it exhibits. However, under real experimental conditions the fitting and prediction performance of a model depend upon its ability to compensate via the regression procedure the various nonidealities, like deviations from the linearity of the pH-gradient profile and the effect of the organic modifier on pK. In general, all models examined exhibit good fitting and prediction performance, and the best model is based on a simple algebraic sigmoid function for 1/k versus pH. However, the main drawback of this model, as well as of all models that are based on the solution of the fundamental equation of gradient elution, is the rather complicated expressions of the retention time. This weakness is overcome by using simple linear models, which give very satisfactory results especially in cases where the retention time varies linearly with the programmed gradient time. The extension of these models so that they are not subject to this restriction is also considered.

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The analytical solutions of the fundamental equation of the multilinear gradient elution are derived in two cases, when the dependence of the logarithm of the solute retention (lnk) upon the volume fraction of organic modifier (φ) is a three-parameter logarithmic expression, and when a simple linear relationship between lnk and lnφ is adopted. The derived theoretical expressions for retention times under multilinear gradient conditions are embodied to simple algorithms for fitting gradient data and especially for resolution optimization. Their performance was examined by using a mixture of 16 model compounds chosen among purines, pyrimidine and nucleosides in eluting systems modified by acetonitrile. It was found that the accuracy of the predicted gradient retention times is very satisfactory even if the simple logarithmic expression for the retention behavior of solutes, i.e. the linear dependence of lnk upon lnφ, is used.

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Four retention models for the effect of aliphatic alcohol additives on the retention of analytes in reversed-phase liquid chromatography have been developed following either a semi-thermodynamic treatment or an empirical approach. Their performance was tested using the experimental retention times of six non-polar analytes (alkylbenzenes) and ten o-phthalaldehyde derivatives of amino acids under different isocratic chromatographic runs when a small amount of ethanol, 1-propanol, 1-butanol, 1-pentanol, 1-hexanol or 1-heptanol was added to methanol/water mixtures containing a constant amount of methanol. It was shown that for the structurally simple alkylbenzenes all the models can be adopted for retention prediction with good results. In contrast, just one out of four models, that with the fewest approximations, predicts satisfactorily the retention properties of amino acids derivatives. However, the most interesting feature is that this model can predict the effect of an alcohol-additive on the retention properties of solutes, even if this additive was not used in chromatographic runs done for the fitting procedure, provided that it belongs to the same homologous series of alkanols. This feature is also observed in all models described the retention of alkylbenzenes.

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The theory of multimode gradient elution in liquid chromatography involving combined gradients of the mobile-phase composition with flow rate and column temperature is presented, and a very simple stepwise method that allows for the calculation of the elution time of a sample solute under all gradient conditions is proposed. The theory is successfully applied to the separation of 12 o-phthalaldehyde derivatives of amino acids in eluting systems modified by acetonitrile. Average errors below 2.9% have been found in the retention prediction using the above method, which is supported by adequate models and algorithms capable of describing the chromatographic behavior of solutes upon changes in the separation factors, such as the modifier content, flow rate, and temperature.

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One- and multi-variable retention models proposed for isocratic and/or gradient elution in reversed-phase liquid chromatography are critically reviewed. The thermodynamic, exo-thermodynamic or empirical arguments adopted for their derivation are presented and discussed. Their connection to the retention mechanism is also indicated and the assumptions and approximations involved in their derivation are stressed. Special attention is devoted to the fitting performance of the various models and its impact on the final predicted error between experimental and calculated retention times. The possibility of using exo-thermodynamic retention models for prediction under gradient elution is considered from a practical point of view. Finally, the use of statistical weights in the fitting procedure of a retention model and its effect on the calculated elution times as well as the transferability of retention data among isocratic and gradient elution modes are also examined and discussed.

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The theory of the dual-mode gradient elution in liquid chromatography involving any type of simultaneous changes in column temperature and mobile-phase composition is developed following Drake's approach. The theory was tested in the retention prediction of six alkylbenzenes in aqueous eluting systems modified by acetonitrile. Significant delay phenomena, i.e., a lag between the programmed gradient temperature and the temperature in the oven, and a lag between the oven temperature and the effective temperature the analyte feels in the column, were detected. When these phenomena were taken into account, the retention prediction obtained for all solutes under all dual-mode gradient conditions was excellent. The average percentage error between experimental and predicted retention times is below to 2%.