Phytochemical Characterization of Rhus coriaria L. Extracts by Headspace Solid-Phase Micro Extraction Gas Chromatography, Comprehensive Two-Dimensional Liquid Chromatography, and Antioxidant Activity Evaluation.

Rhus coriaria L. (Anacardiaceae), commonly known as sumac, has been used since ancient times for many different applications, and nowadays is used mostly as a spice obtained from its in the Mediterranean and the Middle ground fruits and employed for flavoring and garnishing food, predominantly Eastern regions. Traditionally, sumac has been also used in popular medicine for the treatment of many ailments including hemorrhoids, wound healing, diarrhea, ulcers, and eye inflammation. Sumac drupes are indeed rich in various classes of phytochemicals including organic acids, flavonoids, tannins, and others, which are responsible of their powerful antioxidant capacity, from which treatment of many common diseases such as cardiovascular disease, diabetes, and cancer could benefit. In this work we evaluated the influence of fruit ripeness, conservation, and processing. To this aim, a phytochemical characterization of six different samples of Rhus coriaria L. was carried out. Specifically, headspace solid-phase micro extraction gas chromatography coupled to mass spectrometry and comprehensive two-dimensional liquid chromatography coupled to photodiode array and mass spectrometry detection, were employed. A total of 263 volatile compounds, including terpene hydrocarbons, acids, and aldehydes, as well as 83 polyphenolic compounds, mainly gallic acid derivatives, were positively identified. All samples showed a significant antioxidant activity by means of oxygen radical absorbance capacity, in line with their polyphenolic content and composition. Such findings set a solid ground to support the utilization of this plant as an attractive target for novel nutraceutical approaches and for drug discovery.

Polyphenolic compounds with biological activity in guabiroba fruits (Campomanesia xanthocarpa Berg.) by comprehensive two-dimensional liquid chromatography.

This study aimed to evaluate the polyphenolic composition along with the biological activity of guabiroba (Campomanesia xanthocarpa Berg.) fruits using comprehensive two-dimensional liquid chromatography (LC × LC). A simplex centroid design comprising three solvents (methanol, 2% acetic acid, and acetonitrile) was used to optimize the extraction mixture for polyphenols from ripe and unripe guabiroba fruits. A quantitative LC × LC platform was proposed to characterize the guabiroba extracts using a RP-Amide column and a C18 column in the first and second dimensions, respectively. Antidiabetic properties, using in vitro enzyme assay models and in vivo antioxidant activity with the eukaryote model Saccharomyces cerevisiae, was measured. Total phenolics compounds were more efficiently extracted with 2% acetic acid solution and acetonitrile (50:50, v/v). A total of 37 different compounds were identified and quantified using the proposed LC × LC method (linearity ranging from 0.9990 to 0.9994, intra- and interday precision from 0.40 to 10.57% and, accuracy from 81.89 to 108.98%). Significant differences were observed between ripe and unripe guabiroba fruits, especially for the compounds geraldone and methyl galangin isomer. Guabiroba fruits showed significant antidiabetic and antioxidant properties and may be potentially adopted as part of dietary strategies in the management of early stages of type 2 diabetes and associated complications.

Application of compressed fluid-based extraction and purification procedures to obtain astaxanthin-enriched extracts from Haematococcus pluvialis and characterization by comprehensive two-dimensional liquid chromatography coupled to mass spectrometry.

The green microalga Haematococcus pluvialis has been widely studied due to its capacity to accumulate great amounts of astaxanthin, a high-value carotenoid with biological activities. In the present work, two green compressed fluid-based processes, pressurized liquid extraction (PLE) and supercritical antisolvent fractionation (SAF), are integrated to obtain an astaxanthin-enriched extract from this microalga. PLE was carried out using pressurized ethanol as solvent, for 20 min, at 10 MPa, and 50 °C as extraction temperature. Subsequently, the obtained extract was processed by SAF to further purify the carotenoid fraction. The SAF process was optimized using a 3-level factorial experimental design and considering three experimental variables: (i) CO2 pressure (10-30 MPa), (ii) percentage of water in the PLE extract (20-50%), and (iii) PLE extract/supercritical-CO2 flow rate ratio (0.0125-0.05). Total carotenoid content was evaluated in both extracts and raffinates. Best results were obtained at 30 MPa, 0.05 feed/SC-CO2 mass flow rate, and 20% (v/v) of water in the feed solution, achieving values of 120.3 mg g-1 carotenoids in extract (in the SAF extract fraction), which were significantly higher than those obtained in the original PLE extract. In parallel, a new fast two-dimensional comprehensive liquid chromatography (LC×LC) method was optimized to get the full carotenoid profile of these extracts in less than 25 min. This is the first time that the use of a C30 column is reported in an on-line LC×LC system. Graphical abstract.

Heart-cut achiral-chiral LC-LC method development using factorial design: application to the chiral separation of ketoprofen.

A two-dimensional achiral-chiral LC-LC method in heart-cut mode for ketoprofen and its enantiomeric fraction determination was proposed. A C8 column was used in the first dimension, and the chiral column was an α1-acid glycoprotein. The mobile phase of the chiral system was optimized by a factorial design. The effect of temperature on retention and on enantiomeric resolution was studied. Particular attention was paid to mobile phase compatibility for the two columns and to transferring time, using ketoprofen standards. The R-(-) and S-(+)-ketoprofen retention times were 9 and 11 min, respectively; the resolution was higher than 1.1 and enantiomeric fraction close to 0.5. The method was applied to capsules and gels containing ketoprofen. Factorial design was also used to establish the best conditions for gel sample preparation. Recoveries were 84 and 105 % for capsules and gels, respectively. Graphical abstract Two-dimensional chromatogram for KPF and its enantiomers.

A novel four-dimensional analytical approach for analysis of complex samples.

A two-dimensional LC (2D-LC) method, based on the work of Erni and Frei in 1978, was developed and coupled to an ion mobility-high-resolution mass spectrometer (IM-MS), which enabled the separation of complex samples in four dimensions (2D-LC, ion mobility spectrometry (IMS), and mass spectrometry (MS)). This approach works as a continuous multiheart-cutting LC system, using a long modulation time of 4 min, which allows the complete transfer of most of the first - dimension peaks to the second - dimension column without fractionation, in comparison to comprehensive two-dimensional liquid chromatography. Hence, each compound delivers only one peak in the second dimension, which simplifies the data handling even when ion mobility spectrometry as a third and mass spectrometry as a fourth dimension are introduced. The analysis of a plant extract from Ginkgo biloba shows the separation power of this four-dimensional separation method with a calculated total peak capacity of more than 8700. Furthermore, the advantage of ion mobility for characterizing unknown compounds by their collision cross section (CCS) and accurate mass in a non-target approach is shown for different matrices like plant extracts and coffee. Graphical abstract Principle of the four-dimensional separation.

Contaminant screening of wastewater with HPLC-IM-qTOF-MS and LC+LC-IM-qTOF-MS using a CCS database.

Non-target analysis has become an important tool in the field of water analysis since a broad variety of pollutants from different sources are released to the water cycle. For identification of compounds in such complex samples, liquid chromatography coupled to high resolution mass spectrometry are often used. The introduction of ion mobility spectrometry provides an additional separation dimension and allows determining collision cross sections (CCS) of the analytes as a further physicochemical constant supporting the identification. A CCS database with more than 500 standard substances including drug-like compounds and pesticides was used for CCS data base search in this work. A non-target analysis of a wastewater sample was initially performed with high performance liquid chromatography (HPLC) coupled to an ion mobility-quadrupole-time of flight mass spectrometer (IM-qTOF-MS). A database search including exact mass (±5 ppm) and CCS (±1 %) delivered 22 different compounds. Furthermore, the same sample was analyzed with a two-dimensional LC method, called LC+LC, developed in our group for the coupling to IM-qTOF-MS. This four dimensional separation platform revealed 53 different compounds, identified over exact mass and CCS, in the examined wastewater sample. It is demonstrated that the CCS database can also help to distinguish between isobaric structures exemplified for cyclophosphamide and ifosfamide. Graphical Abstract Scheme of sample analysis and database screening.

Detailed comparison of in-house developed and commercially available heart-cutting and selective comprehensive two-dimensional liquid chromatography systems.

Recently, two-dimensional liquid chromatography (2D-LC) has become a popular approach to analyze complex samples. This is partly due to the introduction of commercial 2D-LC systems. In the past, 2D-LC was carried out on in-house developed setups, typically consisting of several switching valves and sample loops as the interface between the two dimensions. Commercial systems usually offer different 2D-LC modes in combination with specialized software to operate the instrument and analyze the data. This makes them highly user-friendly, however, at an increased cost compared to in-house developed setups. This study aims to make a comparison between an in-house developed 2D-LC setup and a commercially available 2D-LC instrument. The comparison is made based on experimental differences, in addition to more general differences, including cost price, flexibility, and ease of operation. Special attention is also paid to the different strategies to deal with the mobile phase incompatibility between the highly orthogonal separation mechanisms considered in this work: hydrophilic interaction liquid chromatography (HILIC) and reversed-phase LC (RPLC). For the commercial 2D-LC instrument, this is done using active solvent modulation (ASM), a valve-based approach allowing the on-line dilution of the effluent eluting from the first dimension column before transfer to the second dimension (2D) column. For the in-house developed setup, a combination of restriction capillaries and a trap column is used. Using a sample of 28 compounds with a large polarity range, peak shapes and recoveries of the 2D-chromatograms are compared for both setups. For early eluting compounds, the selective comprehensive approach, currently only possible on the commercial 2D-LC instrument, results in the best peak shapes and recoveries, however, at the cost of an increased analysis time. In general, depending on the analytical goal (single heart-cut versus full-comprehensive 2D-LC), an in-house developed system can be satisfactory for the analysis of specific target compounds/samples. For more complex problems, it can be interesting to use a more specialized commercial 2D-LC instrument. Overall, this comparison study provides advice for analytical scientists, who are considering to use 2D-LC, on the type of equipment to consider, depending on the needs of their particular applications.

High Step-Up Ratio DC-AC Converter Using Fourth-Order LCLC Resonant Circuit for Ultrasonic Fingerprint-Sensor Drivers.

Ultrasonic fingerprint sensors are becoming more widely used in thick or flexible displays. In order to better identify fingerprint information, ultrasonic sensors need to generate more ultrasonic energy, which can be transmitted to the display surface through media with higher acoustic impedance. In this paper, a DC-AC converter with a high lift ratio was proposed to enhance the transmission energy of the ultrasonic fingerprint sensor, thus helping to improve the identification. The converter comprises a full-bridge inverter and two LC resonant circuits. The introduction of an additional LC resonant circuit into the traditional Class-D LC resonant converter effectively increases the boost ratio of the proposed converter from 5 to 22. When used as a part of the ultrasonic fingerprint sensing system, the proposed converter can amplify the 20-V low-voltage DC required to drive the piezoelectric organic film to 376 V high-voltage AC. The voltage of the wave received from this new driver is equal to 970 mV, which greatly exceeds the 376 mV achieved by using the Class-D converter alone. In this paper, the topology proposed by the ultrasonic fingerprint sensor converter driver was experimentally verified, which greatly improved the boost ratio and can be considered suitable for wider applications.

Evolution of anthocyanin content during grape ripening and characterization of the phenolic profile of the resulting wine by comprehensive two-dimensional liquid chromatography.

The typical phenolic profile in grapes is characterized by its complexity both in terms of number of diverse chemical structures and their variation during ripening. Besides, the specific phenolic composition of grapes directly influences the presence of those components in the resulting wine. In this contribution, a new method based on the application of comprehensive two-dimensional liquid chromatography coupled to a diode array detector and tandem mass spectrometry has been developed to obtain the typical phenolic profile of Malbec grapes cultivated in Brazil. Moreover, the method has been demonstrated to be useful to study how the phenolic composition in grapes evolved during a 10-week ripening period. Main detected compounds in grapes and in the wine derived from them were anthocyanins, although a good number of polymeric flavan-3-ols were also tentatively identified, among other compounds. Results show how the amount of anthocyanins present in grapes was increased during ripening up to 5-6 weeks and then decreased towards week 9. The two-dimensional approach applied was demonstrated to be useful for the characterization of the complex phenolic profile of these samples, involving more than 40 different structures and has the potential to be further applied to the study of this important fraction is different grapes and wines systematically.

A detailed evaluation of the advantages and limitations of online RP-LC×HILIC compared to HILIC×RP-LC for phenolic analysis.

The combination of hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography (RP-LC) has proved effective in the LC × LC analysis of polyphenols due to the high degree of orthogonality associated with these separation modes for various classes of phenolic compounds. However, despite the growing number of such applications, HILIC is almost exclusively used as the first dimension (1D) separation mode, and RP-LC in the second dimension (2D). This is somewhat surprising in light of the potential advantages of swapping these separation modes. In this contribution, we present a detailed evaluation of the potential of online RP-LC × HILIC-MS for the analysis of phenolic compounds, comparing the performance of this system to the more established HILIC × RP-LC-MS configuration. Method development was performed using a predictive optimisation program, and fixed solvent modulation was employed to combat the solvent incompatibility between HILIC and RP-LC mobile phases. Red wine, rooibos tea, Protea and chestnut phenolic extracts containing a large diversity of phenolic compound classes were analysed by both HILIC × RP-LC- and RP-LC × HILIC-MS in order to compare the separation performance. Overall, the kinetic performance of HILIC × RP-LC was found to be clearly superior, with higher peak capacities and better resolution obtained for the majority of samples compared to RP-LC × HILIC analyses using similar column dimensions. Dilution of the 1D solvent combined with large volume injections proved insufficient to focus especially phenolic acids in the 2D HILIC separation, which resulted in severe 2D peak distortion for these compounds, and negatively impacted on method performance. On the other hand, a noteworthy improvement in the sensitivity of RP-LC × HILIC-MS analyses was observed due to higher ESI-MS response for the 2D HILIC mobile phase and greater sample loading capacity of the 1D RP-LC column, brought on by the high solubility of phenolic samples in aqueous solutions. As a result, a significantly higher number of compounds were detected in the RP-LC × HILIC-MS separations. These findings point to the potential advantage of RP-LC × HILIC as a complementary configuration to HILIC × RP-LC for phenolic analysis.

Quantification strategies for two-dimensional liquid chromatography datasets using regions of interest and multivariate curve resolution approaches.

In this work, three chemometrics-based approaches are compared for quantification purposes when using two-dimensional liquid chromatography (LC×LC-MS), taking as a study case the quantification of amino acids in commercial drug mixtures. Although the approaches have been already used for one-dimensional gas or liquid chromatography, the main novelty of this work is the demonstration of their applicability to LC×LC-MS datasets. Besides, steps such as peak alignment and modelling, commonly applied in this type of data analysis, are not required with the approaches proposed here. In a first step, regions of interest (ROI) strategy is used for the spectral compression of the LC×LC-MS datasets. Then the first strategy consists of building a calibration curve from the areas obtained in this ROI compression step. Alternatively, the ROI intensity matrices can be used as input for a second analysis step employing the multivariate curve resolution alternating least squares (MCR-ALS) method. The main benefit of MCR-ALS is the resolution of elution and spectral profiles for each of the analytes in the mixture, even in the case of strong coelutions and high signal overlapping. Classical MCR-ALS based calibration curve from the peak areas resolved only applying non-negativity constraints (second strategy) is compared to the results obtained when an area correlation constraint is imposed during the ALS optimization (third strategy). All in all, similar quantification results were achieved by the three approaches but, especially in prediction studies, the more accurate quantification is obtained when the calibration curve is built from the peak areas obtained with MCR-ALS when the area correlation constraint is imposed.

CIGB-300 Anticancer Peptide Differentially Interacts with CK2 Subunits and Regulates Specific Signaling Mediators in a Highly Sensitive Large Cell Lung Carcinoma Cell Model.

Large cell lung carcinoma (LCLC) is one form of NSCLC that spreads more aggressively than some other forms, and it represents an unmet medical need. Here, we investigated for the first time the effect of the anti-CK2 CIGB-300 peptide in NCI-H460 cells as an LCLC model. NCI-H460 cells were highly sensitive toward CIGB-300 cytotoxicity, reaching a peak of apoptosis at 6 h. Moreover, CIGB-300 slightly impaired the cell cycle of NCI-H460 cells. The CIGB-300 interactomics profile revealed in more than 300 proteins that many of them participated in biological processes relevant in cancer. Interrogation of the CK2 subunits targeting by CIGB-300 indicated the higher binding of the peptide to the CK2α' catalytic subunit by in vivo pull-down assays plus immunoblotting analysis and confocal microscopy. The down-regulation of both phosphorylation and protein levels of the ribonuclear protein S6 (RPS6) was observed 48 h post treatment. Altogether, we have found that NCI-H460 cells are the most CIGB-300-sensitive solid tumor cell line described so far, and also, the findings we provide here uncover novel features linked to CK2 targeting by the CIGB-300 anticancer peptide.

Comprehensive profiling of conjugated fatty acid isomers and their lipid oxidation products by two-dimensional chiral RP×RP liquid chromatography hyphenated to UV- and SWATH-MS-detection.

This research reports on the development of a comprehensive two-dimensional liquid chromatography (2D-LC) method hyphenated to inline DAD-UV and ESI-QTOF-MS/MS-detection for the separation of conjugated polyunsaturated fatty acid isomers and structurally related (saturated, unconjugated, oxidized) compounds. In pharmaceutical lipid formulations conjugated fatty acids can be found as impurities, generated by oxidation of polyunsaturated fatty acids. Due to the structural complexity of resultant multi-component samples one dimensional liquid chromatography may be suboptimal for quality control and impurity profiling. The screened reversed-phase columns showed a lack of selectivity for the conjugated fatty acid isomers but the resolutions improved with the shape selectivity of the stationary phases (C18- < C30- < cholesteryl-ether-bonded). Further enhanced selectivity for the non-chiral conjugated FAs could be achieved with amylose/cellulose-based chiral stationary phases (CSPs) which harbor cavities for selective inclusion depending on E/Z configurations of the double bonds of the analytes. Amylose-based CSPs showed higher selectivity for conjugated fatty acids than the cellulose-based polysaccharide CSPs. Hyphenating the chiral and reversed-phase columns in a comprehensive 2D-LC-setup was favorable since they showed orthogonality and good compatibility, because both were operated under RP-conditions. The chiral dimension (1D) mainly separated the different isomers, while the reversed-phase dimension (2D) separated according to number of double bonds and degree of oxidation. Using this setup, advanced structural annotation of unknowns was possible based on UV-, MS1- and MS2-spectra. Data-independent acquisition (by SWATH) enabled differentiation of positional isomers of oxidized lipids by characteristic MS2-fragments and elucidation of co-eluted compounds by selective extracted ion chromatograms of fragment ions (MS2 EICs).

Using multiple heart-cutting valves and stop-flow operation to enable variable second dimension gradient times in online comprehensive two-dimensional liquid chromatography.

Online comprehensive two-dimensional liquid chromatography (LC×LC) is the preferred method currently for the separation of highly complex mixtures of non-volatile compounds. With fully automated commercial instrumentation and software making the method appealing to both researchers and industry, the demand for systems with improved separation capabilities for highly complex mixtures such as those found in natural products or proteomics has increased. In this study we report an approach that enables variable second dimension analysis times based on the use of multiple heart-cutting valves and stop-flow operation to circumvent the requirement for very fast second dimension (2D) analyses in online LC×LC. As application, the HILIC×RP-LC analysis of condensed tannins (proanthocyanidins) in cocoa, grape seed and quebracho extracts is used to demonstrate the performance on the proposed methodology. The method offers increased flexibility compared to conventional online LC×LC separations in that longer 2D gradients can be used to accommodate more complex portions of the chromatogram, while shorter 2D gradients can be used in sections containing fewer peaks, while largely maintaining the benefits of comprehensive separation. We present an evaluation of the performance of the variable gradient time stop-flow HILIC×RP-LC method compared to a comparable, conventional online HILIC×RP-LC system in terms of practical peak capacities using established 2D-LC theory. The improved separation of especially low-level intermediate molecular weight proanthocyanidin oligomers by the former method demonstrates the benefits of the developed approach.

Comprehensive two-dimensional temperature-responsive × reversed phase liquid chromatography for the analysis of wine phenolics.

Phenolic compounds are an interesting class of natural products because of their proposed contribution to health benefits of foods and beverages and as a bio-source of organic (aromatic) building blocks. Phenolic extracts from natural products are often highly complex and contain compounds covering a broad range in molecular properties. While many 1D-LC and mass spectrometric approaches have been proposed for the analysis of phenolics, this complexity inevitably leads to challenging identification and purification. New insights into the composition of phenolic extracts can be obtained through online comprehensive two-dimensional liquid chromatography (LC × LC) coupled to photodiode array and mass spectrometric detection. However, several practical hurdles must be overcome to achieve high peak capacities and to obtain robust methods with this technique. In many LC × LC configurations, refocusing of analytes at the head of the 2D column is hindered by the high eluotropic strength of the solvent transferred from the 1D to the 2D, leading to peak breakthrough or broadening. LC × LC combinations whereby a purely aqueous mobile phase is used in the 1D and RPLC is used in the 2D are unaffected by these phenomena, leading to more robust methods. In this contribution, the combination of temperature-responsive liquid chromatography (TRLC) with RPLC is used for the first time for the analysis of phenolic extracts of natural origin to illustrate the potential of this alternative combination for natural product analyses. The possibilities of the combination are investigated through analysis of wine extracts by TRLC × RPLC-DAD and TRLC × RPLC-ESI-MS.

Whole-genome characterization of large-cell lung carcinoma: A comparative analysis based on the histological classification.

Background: According to the 2015 World Health Organization classification, large cell neuroendocrine carcinoma (LCNEC) was isolated from Large-cell lung cancer (LCLC) tumors, which constitutes 2%-3% of non-small cell lung cancer (NSCLC). However, LCLC tumors are still fairly vaguely defined at the molecular level compared to other subgroups. Materials and Methods: In this study, whole-genome sequencing (WGS) was performed on 23 LCLC and 15 LCNEC tumor specimens. Meanwhile, data from the TCGA (586 LUADs and 511 LUSCs) and U Cologne (120 SCLCs) were analyzed and compared. Results: The most common driver mutations were found in TP53 (13/23, 57%), FAM135B (8/23, 35%) and FAT3 (7/23, 30%) in LCLC, while their counterparts in LCNEC were TP53 (13/15, 87%), LRP1B (6/15, 40%) and FAT1 (6/15, 40%). Notably, FAM135B mutations only occurred in LCLC (P = 0.013). Cosmic signature analysis revealed widespread defective DNA mismatch repair and tobacco-induced mutations in both LCLC and LCNEC. Additionally, LCNEC had a higher incidence of chromosomal copy number variations (CNVs) and structural variations (SVs) compared with LCLC, although the differences were not statistically significant. Particularly, chromothripsis SVs was significantly associated with CNVs. Furthermore, mutational landscape of different subtypes indicated differences between subtypes, and there seems to be more commonalty between our cohort and SCLC than with other subtypes. SMARCA4 mutations may be specific driver gene alteration in our cohort. Conclusion: Our results support that LCLC and LCNEC tumors follow distinct tumorigenic pathways. To our knowledge, this is the first genome-wide profiling comparison of LCLC and LCNEC.

Bayesian optimization of comprehensive two-dimensional liquid chromatography separations.

Comprehensive two-dimensional liquid chromatography (LC×LC), is a powerful, emerging separation technique in analytical chemistry. However, as many instrumental parameters need to be tuned, the technique is troubled by lengthy method development. To speed up this process, we applied a Bayesian optimization algorithm. The algorithm can optimize LC×LC method parameters by maximizing a novel chromatographic response function based on the concept of connected components of a graph. The algorithm was benchmarked against a grid search (11,664 experiments) and a random search algorithm on the optimization of eight gradient parameters for four different samples of 50 compounds. The worst-case performance of the algorithm was investigated by repeating the optimization loop for 100 experiments with random starting experiments and seeds. Given an optimization budget of 100 experiments, the Bayesian optimization algorithm generally outperformed the random search and often improved upon the grid search. Moreover, the Bayesian optimization algorithm offered a considerably more sample-efficient alternative to grid searches, as it found similar optima to the grid search in far fewer experiments (a factor of 16-100 times less). This could likely be further improved by a more informed choice of the initialization experiments, which could be provided by the analyst's experience or smarter selection procedures. The algorithm allows for expansion to other method parameters (e.g., temperature, flow rate, etc.) and unlocks closed-loop automated method development.

A nomogram prognostic model for large cell lung cancer: analysis from the Surveillance, Epidemiology and End Results Database.

BACKGROUND: Currently, there is no reliable method for predicting the prognosis of patients with large cell lung cancer (LCLC). The aim of this study was to develop and validate a nomogram model for accurately predicting the prognosis of patients with LCLC. METHODS: LCLC patients, diagnosed from 2007 to 2009, were identified from the Surveillance, Epidemiology and End Results (SEER) database and used as the training dataset. Significant clinicopathologic variables (P<0.05) in a multivariate Cox regression were selected to build the nomogram. The performance of the nomogram model was evaluated by the concordance index (C-index), the area under the curve (AUC), and internal calibration. LCLC patients diagnosed from 2010 to 2016 in the SEER database were selected as a testing dataset for external validation. The nomogram model was also compared with the currently used American Joint Committee on Cancer (AJCC) tumor-node-metastasis (TNM) staging system (8th edition) by using C-index and a decision curve analysis. RESULTS: Eight variables-age, sex, race, marital status, T stage, N stage, M stage, and treatment strategy-were statistically significant in the multivariate Cox model and were selected to develop the nomogram model. This model exhibited excellent predictive performance. The C-index and AUC value were 0.761 [95% confidence interval (CI), 0.754 to 0.768] and 0.886 for the training dataset and 0.773 (95% CI, 0.765 to 0.781) and 0.876 for the testing dataset, respectively. This model also predicted three-year and five-year lung cancer-specific survival (LCSS) in both datasets with good fidelity. This nomogram model performs significantly better than the 8th edition AJCC TNM staging system, with a higher C-index (P<0.001) and better net benefits in predicting LCSS in LCLC patients. CONCLUSIONS: We developed and validated a prognostic nomogram model for predicting 3- and 5-year LCSS in LCLC patients with good discrimination and calibration abilities. The nomogram may be useful in assisting clinicians to make individualized decisions for appropriate treatment in LCLC.

Sensory-Guided Multidimensional Exploration of Antisweet Principles from Gymnema sylvestre (Retz) Schult.

We report on activity-guided investigation of the key antisweet principles of Gymnema sylvestre. Orosensory-guided fractionation by means of solid phase extraction, preparative 2D-LC, and semipreparative HPLC followed by accurate MS and 1D/2D NMR experiments revealed six known and three previously unknown gymnemic acids as the key constituents of seven highly sensory-active fractions. Localized via a modified comparative taste dilution analysis (cTDA) and taste modulation probability (TMP) based screening techniques, a strong intrinsic bitterness was also observed for gymnemic acids. In addition, the suppressive effects of the most abundant acids on the response of the human sweet taste receptor to sucrose were verified by means of a functional hTAS1R2/hTAS1R3 sweet taste receptor assay. This in vitro screening revealed large differences in antisweet activity among the isolated compounds, where gymnemic acids XV and XIX showed the highest sweet suppressing activity. This broad-based molecular characterization of the sweet taste inhibiting activity of Gymnema sylvestre will enable further insight into the molecular basis of sweet taste modulation at the receptor level.

DNA-PK inhibition by M3814 enhances chemosensitivity in non-small cell lung cancer.

A significant proportion of non-small cell lung cancer (NSCLC) patients experience accumulating chemotherapy-related adverse events, motivating the design of chemosensitizating strategies. The main cytotoxic damage induced by chemotherapeutic agents is DNA double-strand breaks (DSB). It is thus conceivable that DNA-dependent protein kinase (DNA-PK) inhibitors which attenuate DNA repair would enhance the anti-tumor effect of chemotherapy. The present study aims to systematically evaluate the efficacy and safety of a novel DNA-PK inhibitor M3814 in synergy with chemotherapies on NSCLC. We identified increased expression of DNA-PK in human NSCLC tissues which was associated with poor prognosis. M3814 potentiated the anti-tumor effect of paclitaxel and etoposide in A549, H460 and H1703 NSCLC cell lines. In the four combinations based on two NSCLC xenograft models and two chemotherapy, we also observed tumor regression at tolerated doses in vivo. Moreover, we identified a P53-dependent accelerated senescence response by M3814 following treatment with paclitaxel/etoposide. The present study provides a theoretical basis for the use of M3814 in combination with paclitaxel and etoposide in clinical practice, with hope to aid the optimization of NSCLC treatment.