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Characterization of carbohydrate structures using mass spectrometry is a challenging task. Understanding the dissociation mechanisms of carbohydrates in the gas phase is crucial for characterizing these structures through tandem mass spectrometry. In this study, we investigated the collision-induced dissociation (CID) of glucose, galactose, and mannose in their linear forms, as well as the linear forms of hexose at the reducing end of 1-6 linked disaccharides, using quantum chemistry calculations and tandem mass spectrometry. Our results suggest that the dehydration reaction in linear structures is unlikely to occur due to the significantly high reaction barrier compared to those of C═O migration and C-C bond cleavage. We demonstrate that the different intensities of the cross-ring fragments observed in the CID spectra can be explained by the different transition state energies of C═O migration and C2-C3, C3-C4, and C4-C5 bond cleavages, and the branching ratios of the cross-ring fragments are significantly different between glucose and galactose. The application of the cross-ring fragments to oligosaccharides reveals that the stereoisomers of glucose and galactose in oligosaccharides can be differentiated based on the relative intensities of the cross-ring fragments produced by the C2-C3 bond cleavage and C3-C4 bond cleavage, a differentiation that cannot be achieved by conventional tandem mass spectrometry.

Fructooligosaccharides (FOS) and raffinose family oligosaccharides (RFOs) are two highly abundant water-soluble carbohydrates in plants. The typical procedures for the FOS and RFO structural determination using mass spectrometry involve permethylation, followed by the hydrolysis of the permethylated oligosaccharides into monosaccharides, and then the identification of linkage positions using GC mass spectrometry. However, the determination of linkage position sequence is not straightforward, thus this method is limited to small oligosaccharides or oligosaccharides with simple linkages. In this study, we employed a new mass spectrometry method, logically derived sequence tandem mass spectrometry, to determine the structures of FOS and RFOs. We first showed that the monosaccharide and disaccharide CID spectra of aldohexose and ketohexose can be rationalized using dissociation mechanisms. Then we demonstrated that the linkage positions of FOS and RFOs can be identified, the sequence of the linkages can be determined, and the ketohexose and aldohexose in FOS and RFOs can be differentiated, suggesting this new method is useful for structural determination of FOS and RFOs.

High mannose N -glycans extracted from eight different beans (black bean, soybean, pea, white kidney bean, pinto bean, mung bean, white hyacinth bean, and red bean) were studied using the state-of-the-art mass spectrometry method logically derived sequence tandem mass spectrometry (LODES/MS n ). These beans show very similar N -glycan isomer profiles: one isomer of Man 9 GlcNAc 2 and Man 8 GlcNAc 2 , two isomers of Man 7 GlcNAc 2 , three isomers of Man 6 GlcNAc 2 , and five isomers of Man 5 GlcNAc 2 were found. Isomers not predicted by current N -glycan biosynthetic pathways were found in all beans, indicating the possibility of alternative biosynthetic pathways in these plants. The high abundance of unusual high mannose Man 5 GlcNAc 2 N -glycans in beans is particularly useful for the large-scale preparation of high mannose N -glycans that are not easily found in the other biological systems., Competing Interests: The authors declare the following competing financial interest(s): C.Y.L. and C.K.N. are coinventors of a US patent (US 10796788 B2); a part of the method described in the patent for determining the carbohydrate structure was used in this work. All other authors declare no competing interests., (© 2024 The Authors. Published by American Chemical Society.)

N -Linked glycosylation is one of the most essential post-translational modifications of proteins. However, N -glycan structural determination remains challenging because of the small differences in structures between isomers. In this study, we constructed a database containing collision-induced dissociation MS n mass spectra and chromatograms of high-performance liquid chromatography for the rapid identification of high-mannose and paucimannose N -glycan isomers. These N -glycans include isomers by breaking of arbitrary numbers of glycosidic bonds at arbitrary positions of canonical Man 9 GlcNAc 2 N -glycans. In addition, some GlcMan n GlcNAc 2 N -glycan isomers were included in the database. This database is particularly useful for the identification of the N -glycans not in conventional N -glycan standards. This study demonstrated the application of the database to structural assignment for high-mannose N -glycans extracted from bovine whey proteins, soybean proteins, human mammary epithelial cells, and human breast carcinoma cells. We found many N -glycans that are not expected to be generated by conventional biosynthetic pathways of multicellular eukaryotes.

In recent years, academics have paid more attention to green finance, and public companies have reached a broad consensus on the concept of timely environmental, social, and governance (ESG) disclosure. Due to the close relationship between green finance and ESG, this presents an opportunity to determine whether green finance compels companies to actively disclose ESG. The sample for this study consists of China's non-financial A-share listed companies from 2010 to 2021, and the empirical findings demonstrate that green finance can positively influence the ESG performance of listed companies. Through an analysis of heterogeneity, this study reaches the following conclusions: state-owned enterprises, heavy pollution companies, and companies in low-carbon pilot cities perform better in terms of green finance's role in promoting ESG scoring. This study also introduces market concentration and social trust as the moderating variables, enriching the green finance research framework. Through the analysis of moderating variables, the 'black box' effect of green finance on ESG is disclosed, providing theoretical support for the government and companies to better comprehend the policy effect as well as a reference for reform and experimental promotion of green finance., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Determining carbohydrate structures, such as their compositions, linkage positions, and in particular the anomers and stereoisomers, is a great challenge. Isomers of different anomers or stereoisomers have the same sequences of chemical bonds, but have different orientations of some chemical bonds which are difficult to be distinguished by mass spectrometry. Collision-induced dissociation (CID) tandem mass spectroscopy (MS/MS) is a widely used technique for characterizing carbohydrate structures. Understanding the carbohydrate dissociation mechanism is important for obtaining the structural information from MS/MS. In this work, we studied the CID mechanism of galactose- N -acetylgalactosamine (Gal-GalNAc) and glucose- N -acetylglucosamine (Glc-GlcNAc) disaccharides with 1→3 and 1→4 linkages. For Gal-GalNAc disaccharides, the CID mass spectra of sodium ion adducts show significant difference between the α- and β-anomers of GalNAc at the reducing end, while no difference in the CID mass spectra between two anomers of Glc-GlcNAc disaccharides was found. Quantum chemistry calculations show that for Gal-GalNAc disaccharides, the difference of the dissociation barriers between dehydration and glycosidic bond cleavage is significantly small in the β-anomer compared to that in the α-anomer; while these differences are similar between the α- and β-anomers of Glc-GlcNAc disaccharides. These differences can be attributed to the different orientations of hydroxyl and N -acetyl groups located at GalNAc and GlcNAc. The calculation results are consistent with the CID spectra of isotope labelled disaccharides. Our study provides an insight into the CID of 1→3 and 1→4 linked Gal-GalNAc and Glc-GlcNAc disaccharides. This information is useful for determining the anomeric configurations of GalNAc in oligosaccharides.

N -linked glycosylation is one of the most important post-translational modifications of proteins. Current knowledge of multicellular eukaryote N -glycan biosynthesis suggests high mannose N -glycans are generated in the endoplasmic reticulum and Golgi apparatus through conserved biosynthetic pathways. According to conventional biosynthetic pathways, four Man 7 GlcNAc 2 isomers, three Man 6 GlcNAc 2 isomers, and one Man 5 GlcNAc 2 isomer are generated during this process. In this study, we applied our latest mass spectrometry method, logically derived sequence tandem mass spectrometry (LODES/MS n ), to re-examine high mannose N -glycans extracted from various multicellular eukaryotes which are not glycosylation mutants. LODES/MS n identified many high mannose N -glycan isomers previously unreported in plantae, animalia, cancer cells, and fungi. A database consisting of retention time and CID MS n mass spectra was constructed for all possible Man n GlcNAc 2 ( n = 5, 6, 7) isomers that include the isomers by removing arbitrary numbers and positions of mannose from canonical N -glycan, Man 9 GlcNAc 2 . Many N -glycans in this database are not found in current N -glycan mass spectrum libraries. The database is useful for rapid high mannose N -glycan isomeric identification.

The graph-theoretic based studies employing bipartite network approach mostly focus on surveying the statistical properties of the structure and behavior of the network systems under the domain of complex network analysis. They aim to provide the big-picture-view insights of a networked system by looking into the dynamic interaction and relationship among the vertices. Nonetheless, incorporating the features of individual vertex and capturing the dynamic interaction of the heterogeneous local rules governing each of them in the studies is lacking. The methodology in achieving this could hardly be found. Consequently, this study intends to propose a methodology framework that considers the influence of heterogeneous features of each node to the overall network behavior in modeling real-world bipartite network system. The proposed framework consists of three main stages with principal processes detailed in each stage, and three libraries of techniques to guide the modeling activities. It is iterative and process-oriented in nature and allows future network expansion. Two case studies from the domain of communicable disease in epidemiology and habitat suitability in ecology employing this framework are also presented. The results obtained suggest that the methodology could serve as a generic framework in advancing the current state of the art of bipartite network approach., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2023.)

N-linked glycosylation is one of the most important post translational modification of proteins. Various analytical techniques are used for the structural identification of the N-glycans released from proteins through various enzymatic and chemical methods. Although very few side-reaction products are generated during the enzymatic release of N-glycans, this method is expensive and suitable only for small quantities of samples. By contrast, chemical methods can be used for large quantities of samples; however, various side-reaction products are generated when chemical methods are used. Recently, the ammonia-catalyzed release of N-glycans from proteins has been reported to be associated with no typical side reactions. In the present study, we discovered a new side reaction: the epimerization of N-acetylglucosamine present at the reducing end of N-glycans to N-acetylmannosamine. The product of this side reaction interfered with the structural identification N-glycans. We propose a simple method that can help identify this artifact N-glycan isomer and eliminate the aforementioned interference. This simple method widens the applicability of ammonia-catalyzed reactions for N-glycan release from proteins, and is also suitable for N-glycans released using any other alkaline solutions., Competing Interests: Declarationof competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Collision-induced dissociation (CID) tandem mass spectrometry is commonly used for carbohydrate structural determinations. In the CID tandem mass spectrometry approach, carbohydrates are dissociated into fragments, and this is followed by the structural identification of fragments through subsequent CID. The success of the structural analysis depends on the structural correlation of fragments before and after dissociation, that is, structural memory of fragments. Fragments that completely lose the memory of their original structures cannot be used for structural analysis. By contrast, fragments with extremely strong correlations between the structures before and after fragmentation retain the information on their original structures as well as have memories of their precursors' entire structures. The CID spectra of these fragments depend on their own structures and on the remaining parts of the precursor structures, making structural analysis impractical. For effective structural analysis, the fragments produced from a precursor must have good structural memory, meaning that the structures of these fragments retain their original structure, and they must not be strongly affected by the remaining parts of the precursors. In this study, we found that most of the carbohydrate fragments produced by low-energy CID have good memory in terms of linkage position and anomericity. Fragments with ugly memory, where fragment structures change with the remaining parts of the precursors, can be attributed to C ion formation in a linear form. Fragments with ugly memory can be changed to have good memory by preventing linear C ion generation by using an alternative CID sequence, or the fragments of ugly memory can become useful in structural analysis when the contribution of linear C ions in fragmentation patterns is understood.

Rational: Electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) are soft ionization techniques commonly used in mass spectrometry. Although in-source and post-source decays of MALDI have been investigated extensively, the analogous decays of ESI have received little attention. Previous studies regarding the analogous decays of ESI focus on the dissociation of multiply charged proteins and peptides. The decay of carbohydrates in ESI has not been investigated yet, and it may have interference in carbohydrate structural determination., Methods: Commercial apparatus, including a high-performance liquid chromatography (HPLC), an ESI source, and a linear ion trap mass spectrometer, were used to investigate the fragmentation of several N-glycans during the ESI process., Results: About 0.2%-3% of neutral N-glycans and more than 50% of N-glycans consisting of a sialic acid are dissociated into small N-glycans by ESI in-source decay in typical ESI operating conditions. The efficiencies of most dissociation channels increase as the temperature of ion transfer capillary increases, indicating that part of the energy deposited into the precursor ions for cracking is from the heated capillary. The cracking patterns of ESI in-source decay are slightly different from those of gaseous phase collision-induced dissociation., Conclusions: Large N-glycans are dissociated into small N-glycans in ESI in-source decay that may result in the interference of the structural identification of small N-glycans. Separation of large N-glycans from small N-glycans, for example, using HPLC, prior to ESI ionization is necessary to eliminate the interference. This is particularly important when N-glycans consist of sialic acid or large N-glycans have much higher concentration than that of small N-glycans in ESI solution., (© 2022 John Wiley & Sons Ltd.)

Free oligosaccharides are abundant macronutrients in milk and involved in prebiotic functions and antiadhesive binding of viruses and pathogenic bacteria to colonocytes. Despite the importance of these oligosaccharides, structural determination of oligosaccharides is challenging, and milk oligosaccharide biosynthetic pathways remain unclear. Oligosaccharide structures are conventionally determined using a combination of chemical reactions, exoglycosidase digestion, nuclear magnetic resonance spectroscopy, and mass spectrometry. Most reported free oligosaccharides are highly abundant and have lactose at the reducing end, and current oligosaccharide biosynthetic pathways in human milk are proposed based on these oligosaccharides. In this study, a new mass spectrometry technique, which can identify linkages, anomericities, and stereoisomers, was applied to determine the structures of free oligosaccharides in human, bovine, and caprine milk. Oligosaccharides that do not follow the current biosynthetic pathways and are not synthesized by any discovered enzymes were found, indicating the existence of undiscovered biosynthetic pathways and enzymes., (© 2022. The Author(s).)

Despite the importance of carbohydrates in biological systems, structural determination of carbohydrates remains difficult because of the large number of isomers. In this study, a new mass spectrometry method, namely logically derived sequence tandem mass spectrometry (LODES/MS n ), was developed to characterize oligosaccharide structures. In this approach, sequential collision-induced dissociation (CID) of oligosaccharides is performed in an ion trap mass spectrometer to identify the linkage position, anomeric configuration, and stereoisomers of each monosaccharide in the oligosaccharides. The CID sequences are derived from carbohydrate dissociation mechanisms. LODES/MS n does not require oligosaccharide standards or the prior knowledge of the rules and principles of biosynthetic pathways; thus LODES/MS n is particularly useful for the investigation of undiscovered oligosaccharides. We demonstrated that the structure of core oligosaccharides in glycosphingolipids can be identified from more than 500 000 isomers using LODES/MS n . The same method can be applied for determining the structures of other oligosaccharides, such as N -, and O -glycans, and free oligosaccharides in milk.

N-linked glycosylation is one of the most important protein post-translational modifications. Despite the importance of N-glycans, the structural determination of N-glycan isomers remains challenging. Here we develop a mass spectrometry method, logically derived sequence tandem mass spectrometry (LODES/MS n ), to determine the structures of N-glycan isomers that cannot be determined using conventional mass spectrometry. In LODES/MS n , the sequences of successive collision-induced dissociation are derived from carbohydrate dissociation mechanisms and apply to N-glycans in an ion trap for structural determination. We validate LODES/MS n using synthesized N-glycans and subsequently applied this method to N-glycans extracted from soybean, ovalbumin, and IgY. Our method does not require permethylation, reduction, and labeling of N-glycans, or the mass spectrum databases of oligosaccharides and N-glycan standards. Moreover, it can be applied to all types of N-glycans (high-mannose, hybrid, and complex), as well as the N-glycans degraded from larger N-glycans by any enzyme or acid hydrolysis., (© 2021. The Author(s).)

Mass spectrometry has high sensitivity and is widely used in the identification of molecular structures, however, the structural determination of oligosaccharides through mass spectrometry is still challenging. A novel method, namely the logically derived sequence (LODES) tandem mass spectrometry (MS n ), for the structural determination of underivatized oligosaccharides was developed. This method, which is based on the dissociation mechanisms, involves sequential low-energy collision-induced dissociation (CID) of sodium ion adducts, a logical sequence for identifying the structurally decisive product ions for subsequent CID, and a specially prepared disaccharide CID spectrum database. In this work, we reported the assignment of the specially prepared galactose disaccharide CID spectra. We used galactose trisaccharides and tetrasaccharides as examples to demonstrate LODES/MS n is a general method that can be used for the structural determination of hexose oligosaccharides. LODES/MS n has the potential to be extended to oligosaccharides containing other monosaccharides provided the dissociation mechanisms are understood and the corresponding disaccharide database is available.

Glycans have diverse functions and play vital roles in many biological systems, such as influenza, vaccines, and cancer biomarkers. However, full structural identification of glycans remains challenging. The glycan structure was conventionally determined by chemical methods or NMR spectroscopy, which require a large amount of sample and are not readily applicable for glycans extracted from biological samples. Although it has high sensitivity and is widely used for structural determination of molecules, current mass spectrometry can only reveal parts of the glycan structure. Herein, the full structures of glycans, including diastereomers, the anomericity of each monosaccharide, and the linkage position of each glycosidic bond, which can be determined by using tandem mass spectrometry guided by a logically derived sequence (LODES), are shown. This new method provides de novo oligosaccharide structural identification with high sensitivity and has been applied to automatic in situ structural determination of oligosaccharides eluted by means of HPLC. It is shown that the structure of a given trisaccharide from a trisaccharide mixture and bovine milk were determined from nearly 3000 isomers by using 6-7 logically selected collision-induced dissociation spectra. The entire procedure for mass spectrometry measurement guided by LODES can be programmed in a computer for automatic full glycan structure identification., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Carbohydrates play important roles in biological recognition processes. However, determining the structures of carbohydrates remains challenging because of their complexity. A simple tandem mass spectrometry-based method for determining the structure of underivatized mannose tetrasaccharides was demonstrated. This method employed the multistage low-energy collision-induced dissociation (CID) of sodium adducts in an ion trap, a logically derived sequence (LODES) from the dissociation mechanism for deciding the sequence of CID, and a specially prepared disaccharide spectrum database. Through this method, the linkages, anomeric configurations, and branch locations of carbohydrates could be determined without the prior assumption of possible structures. We validated this method by blind test of all the commercial available mannose tetrasaccharides. We showed that the structure of a given tetrasaccharide can be determined from 928 isomers by using only three to six appropriately selected CID mass spectra according to the proposed procedure. This method is simple and rapid and has the potential to be applied to other hexoses and oligosaccharides larger than tetrasaccharides. The CID procedures can be built in a computer-controlled mass spectrometer for automatic structural determination of underivatized oligosaccharides. Graphical abstract.

A rapid mass spectrometry method for determining the anomeric configuration of the sugar at the reducing end of an oligosaccharide was demonstrated. The method was employed to identify the nascent anomeric configuration (i.e., before significant mutarotation occurs) of oligosaccharides released by carbohydrate-active enzymes, which enabled determination of the enzyme mechanism. This method was validated by applying it to various enzymes, including α-glucosidase, β-glucosidases, endoglycoceramidase II, β-galactosidase, and β-amylase., (Copyright © 2019. Published by Elsevier Ltd.)

Collision-induced dissociation of sodiated α-glucose, β-glucose, α-galactose, β-galactose, α-mannose, and β-mannose was studied using electronic structure calculations and resonance excitation in a low-pressure linear ion trap. We made an extensive search of conformers and transition states in calculations to ensure the transition state with the lowest barrier height for each dissociation channel could be located. The major dissociation channels, in addition to desodiation, are cross-ring dissociation and dehydration. Cross-ring dissociation starts with H atom transfer from the O1 atom to the O0 atom, followed by the cleavage of the C1-O0 bond. Dehydration of the anomer with O1 and O2 atoms in the cis configuration involves the transfer of an H atom from the O2 atom to the O1 atom, followed by the cleavage of the C1-O1 bond. In contrast, dehydration of the anomer with O1 and O2 atoms in the trans configuration mainly occurs through H atom transfer from the O3 or O2 atom to the O1 atom for glucose, from the O3 or O4 atom to the O1 atom for galactose, and from the O4 or O2 atom to the O1 atom for mannose, followed by the cleavage of the C1-O1 bond. The dehydration barrier heights are lower than those of cross-ring dissociation for cis anomers, but higher than those of cross-ring dissociation for trans anomers. The relative barrier heights from calculations are consistent with the experimental measurements of branching ratios. Both computational and experimental results show that the branching ratio of dehydration can be generalized as a simple rule for rapidly identifying the anomeric configurations of these monosaccharides.

Carbohydrates have various functions in biological systems. However, the structural analysis of carbohydrates remains challenging. Most of the commonly used methods involve derivatization of carbohydrates or can only identify part of the structure. Here, we report a de novo method for completely structural identification of underivatised oligosaccharides. This method, which can provide assignments of linkages, anomeric configurations, and branch locations, entails low-energy collision-induced dissociation (CID) of sodium ion adducts that enable the cleavage of selective chemical bonds, a logical procedure to identify structurally decisive fragment ions for subsequent CID, and the specially prepared disaccharide CID spectrum databases. This method was first applied to determine the structures of four underivatised glucose oligosaccharides. Then, high-performance liquid chromatography and a mass spectrometer with a built-in logical procedure were established to demonstrate the capability of the in situ CID spectrum measurement and structural determination of the oligosaccharides in chromatogram. This consolidation provides a simple, rapid, sensitive method for the structural determination of glucose oligosaccharides, and applications to oligosaccharides containing hexoses other than glucose can be made provided the corresponding disaccharide databases are available.

Oligosaccharides have diverse functions in biological systems. However, the structural determination of oligosaccharides remains difficult and has created a bottleneck in carbohydrate research. In this study, a new approach for the de novo structural determination of underivatized oligosaccharides is demonstrated. A low-energy collision-induced dissociation (CID) of sodium ion adducts was used to facilitate the cleavage of desired chemical bonds during the dissociation. The selection of fragments for the subsequent CID was guided using a procedure that we built from the understanding of the saccharide dissociation mechanism. The linkages, anomeric configurations, and branch locations of oligosaccharides were determined by comparing the CID spectra of oligosaccharide with the fragmentation patterns based on the dissociation mechanism and our specially prepared disaccharide CID spectrum database. The usefulness of this method was demonstrated to determine the structures of several mannose trisaccharides. This method can also be applied in the structural determination of oligosaccharides larger than trisaccharides and containing hexose other than mannose if authentic standards are available. Graphical Abstract.

Collision-induced dissociation (CID) of sodiated glucose was investigated using electronic structure calculations and resonance excitation in a low-pressure linear ion trap. The major dissociation channels in addition to desodiation are dehydration and C 2 H 4 O 2 elimination reactions which the barrier heights are near to or lower than the sodiation energy of glucose. Dehydration reaction involves the transfer of the H atom from the O2 atom to the O1 atom, followed by the cleavage of the C1-O1 bond. Notably, α-glucose has a dehydration barrier lower than that of β-glucose. This difference results in the larger branching ratio of dehydration reactions involving α-glucose, which provides a simple and fast method for identifying the anomeric configurations of glucose. The C 2 H 4 O 2 elimination starts from the H atom transfer from the O1 atom to the O0 atom, followed by the cleavage of the C1-O0 bond. These results were further confirmed by experimental study using 18 O-isotope-labeled compounds. Both the experimental data and theoretical calculations suggest that the dehydration reaction and cross-ring dissociation of sodiated carbohydrates mainly occur at the reducing end during low-energy CID.

We have developed a method for analysing vitamin E using ultra-performance convergence chromatography with a chromatographic runtime of 5.5 min. A well-resolved chromatogram with excellent precision in retention time revealed seven vitamin E components in the palm oil derived tocotrienol-rich fraction. The major vitamin E components were α-tocopherol, α-tocotrienol, γ-tocotrienol and δ-tocotrienol whereas the minor vitamin E components were α-tocomonoenol, β-tocotrienol and an unreported trace component. The new component was positively identified by high-resolution mass spectrometry as 2-methyl-2(4',8',12'-trimethyltrideca-7',11'-dienyl)5,7,8-trimethylchroman-6-ol or α-tocodienol., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Aims: The role of thioredoxin reductase (TrxR) in tumorigenesis has made it an attractive anticancer target. A systematic approach for development of novel compounds as TrxR inhibitors is currently lacking. Structurally diversified TrxR inhibitors share in common electrophilic propensities for the sulfhydryl groups, among which include the Michael reaction acceptors containing an α,β-unsaturated carbonyl moiety. We aimed to identify features among structurally diversified Michael acceptor-based compounds that would yield a strong TrxR inhibitory character., Results: Structurally dissimilar Michael acceptor-based natural compounds such as isobutylamides, zerumbone, and shogaols (SGs) were found to possess a poor TrxR inhibitory activity, indicating that a sole Michael acceptor moiety was insufficient to produce TrxR inhibition. The 1,7-diphenyl-hept-3-en-5-one pharmacophore in 3-phenyl-3-SG, a novel SG analog that possessed comparable TrxR inhibitory and antiproliferative potencies as 6-SG, was modified to yield 1,5-diphenyl-pent-1-en-3-one (DPPen) and 1,3-diphenyl-pro-1-en-3-one (DPPro, also known as chalcone) pharmacophores. These Michael acceptor-centric pharmacophores, when substituted with the hydroxyl and fluorine groups, gave rise to analogs displaying a TrxR inhibitory character positively correlated to their antiproliferative potencies. Lead analogs 2,2'-diOH-5,5'-diF-DPPen and 2-OH-5-F-DPPro yielded a half-maximal TrxR inhibitory concentration of 9.1 and 10.5 μM, respectively, after 1-h incubation with recombinant rat TrxR, with the C-terminal selenocysteine residue found to be targeted., Innovation: Identification of Michael acceptor-centric pharmacophores among diversified compounds demonstrates that a systematic approach to discover and develop Michael acceptor-based TrxR inhibitors is feasible., Conclusion: A strong TrxR inhibitory character correlated to the antiproliferative potency is attributed to structural features that include an α,β-unsaturated carbonyl moiety centered in a DPPen or DPPro pharmacophore bearing hydroxyl and fluorine substitutions.

ADMET (absorption, distribution, metabolism, excretion, and toxicity)-related failure of drug candidates is a major issue for the pharmaceutical industry today. Prediction of PD-PK-T properties using in silico tools has become very important in pharmaceutical research to reduce cost and enhance efficiency. PaDEL-DDPredictor is an in silico tool for rapid prediction of PD-PK-T properties of compounds from their chemical structures. It is free and open-source software that, has both graphical user interface and command line interface, can work on all major platforms (Windows, Linux, and MacOS) and supports more than 90 different molecular file formats. The software can be downloaded from http://padel.nus.edu.sg/software/padelddpredictor., (Copyright © 2012 Wiley Periodicals, Inc.)

In this study, the ensemble of features and training samples was examined with a collection of support vector machines. The effects of data sampling methods, ratio of positive to negative compounds, and types of base models combiner to produce ensemble models were explored. The ensemble method was applied to produce four separate in silico models to classify the labels for eye/skin corrosion (H314), skin irritation (H315), serious eye damage (H318), and eye irritation (H319), which are defined in the "Globally Harmonized System of Classification and Labelling of Chemicals". To the best of our knowledge, the training set used in this work is one of the largest (made of publicly available data) with acceptable prediction performances. These models were distributed via PaDEL-DDPredictor (http://padel.nus.edu.sg/software/padelddpredictor) that can be downloaded freely for public use., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Metabolic activation of chemicals into covalently reactive species might lead to toxicological consequences such as tissue necrosis, carcinogenicity, teratogenicity, or immune-mediated toxicities. Early prediction of this undesirable outcome can help in selecting candidates with increased chance of success, thus, reducing attrition at all stages of drug development. The ensemble modelling of mixed features was used for the development of a model to classify the metabolic activation of chemicals into covalently reactive species. The effects of the quality of base classifiers and performance measure for sorting were examined. An ensemble model of 13 naive Bayes classifiers was built from a diverse set of 1,479 compounds. The ensemble model was validated internally with five-fold cross validation and it has achieved sensitivity of 67.4% and specificity of 93.4% when tested on the training set. The final ensemble model was made available for public use.

Drug-induced liver injury, although infrequent, is an important safety concern that can lead to fatality in patients and failure in drug developments. In this study, we have used an ensemble of mixed learning algorithms and mixed features for the development of a model to predict hepatic effects. This robust method is based on the premise that no single learning algorithm is optimum for all modelling problems. An ensemble model of 617 base classifiers was built from a diverse set of 1,087 compounds. The ensemble model was validated internally with five-fold cross-validation and 25 rounds of y-randomization. In the external validation of 120 compounds, the ensemble model had achieved an accuracy of 75.0%, sensitivity of 81.9% and specificity of 64.6%. The model was also able to identify 22 of 23 withdrawn drugs or drugs with black box warning against hepatotoxicity. Dronedarone which is associated with severe liver injuries, announced in a recent FDA drug safety communication, was predicted as hepatotoxic by the ensemble model. It was found that the ensemble model was capable of classifying positive compounds (with hepatic effects) well, but less so on negatives compounds when they were structurally similar. The ensemble model built in this study is made available for public use., (© Springer Science+Business Media B.V. 2011)

White rot fungi are good lignin degraders and have the potential to be used in industry. In the present work, Phellinus sp., Daedalea sp., Trametes versicolor and Pycnoporus coccineus were selected due to their relatively high ligninolytic enzyme activity, and grown on Acacia mangium wood chips under solid state fermentation. Results obtained showed that manganese peroxidase produced is far more compared to lignin peroxidase, suggesting that MnP might be the predominating enzymes causing lignin degradation in Acacia mangium wood chips. Cellulase enzyme assays showed that no significant cellulase activity was detected in the enzyme preparation of T. versicolor and Phellinus sp. This low cellulolytic activity further suggests that these two white rot strains are of more interest in lignin degradation. The results on lignin losses showed 20-30% of lignin breakdown at 60 days of biodegradation. The highest lignin loss was found in Acacia mangium biotreated with T. versicolor after 60 days and recorded 26.9%, corresponding to the percentage of their wood weight loss recorded followed by P. coccineus. In general, lignin degradation was only significant from 20 days onwards. The overall percentage of lignin weight loss was within the range of 1.02-26.90% over the biodegradation periods. Microscopic observations conducted using scanning electron microscope showed that T. versicolor, P. coccineus, Daedalea sp. and Phellinus sp. had caused lignin degradation in Acacia mangium wood chips.

We previously showed that 3-(2-hydroxyphenyl)-1-(5-methyl-furan-2-y-l)propenone (HMP), suppressed the synthesis of various proinflammatory mediators. In this study, HMP showed a dose-dependent inhibition of NO synthesis in the RAW 264.7 murine macrophage line. The inhibition of NO synthesis was related to inhibition of p38 phosphorylation and kinase activity that led to significant inhibition of phosphorylation of ATF-2. This effect in turn caused inhibition of AP-1-DNA binding which partially explains the inhibitory effect upon the synthesis of iNOS. HMP had no effect upon phosphorylation of JNK, ERK1/2 and STAT-1. Kinase activity of JNK and ERK1/2 was also not affected by HMP as determined by levels of phosphorylated c-jun and phosphorylated elk-1. Furthermore HMP failed to block phosphorylation of IκBα, and subsequent nuclear translocation and DNA-binding activity of p65 NF-κB in IFN-γ/LPS-induced RAW 264.7 cells. Molecular docking experiments confirmed that HMP fits well in the highly conserved hydrophobic pocket of p38 MAP kinase. We conclude that the synthetic HMP is a chalcone analogue that selectively inhibits the p38/ATF-2 and AP-1 signaling pathways in the NO synthesis by the macrophage RAW 264.7., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Objectives: We evaluated several flavonoid combinations for synergy in the inhibition of proinflammatory mediator synthesis in the RAW 264.7 cellular model of inflammation., Methods: The inhibitory effect of chrysin, kaempferol, morin, silibinin, quercetin, diosmin and hesperidin upon nitric oxide (NO), prostaglandin E(2) (PGE(2)) and tumour necrosis factor-alpha (TNF-alpha) secretion from the LPS-induced RAW 264.7 monocytic macrophage was assessed and IC(50) values obtained. Flavonoids that showed reasonable inhibitory effects in at least two out of the three assays were combined in a series of fixed IC(50) ratios and reassessed for inhibition of NO, PGE(2) and TNF-alpha. Dose-response curves were generated and interactions were analysed using isobolographic analysis., Results: The experiments showed that only chrysin, kaempferol, morin, and silibinin were potent enough to produce dose-response effects upon at least two out of the three mediators assayed. Combinations of these four flavonoids showed that several combinations afforded highly significant synergistic effects., Conclusions: Some flavonoids are synergistic in their anti-inflammatory effects when combined. In particular chrysin and kaempferol significantly synergised in their inhibitory effect upon NO, PGE(2) and TNF-alpha secretion. These findings open further avenues of research into combinatorial therapeutics of inflammatory-related diseases and the pharmacology of flavonoid synergy.

HMP [3-(2-hydroxyphenyl)-1-(5-methyl-furan-2-y-l) propenone] was evaluated for its ability to inhibit the synthesis of major proinflammatory mediators and cytokines in interferon-gamma (IFN-gamma)- and lipopolysaccharide (LPS)-induced RAW 264.7 cells and phorbol myristate acetate (PMA)-differentiated/LPS-induced U937 cells. HMP suppressed the production of nitric oxide (NO) with significant inhibitory effects at doses as low as 0.78 microM (P < 0.05). Prostaglandin E2 (PGE2) secretion was also inhibited at doses of 12.5 microM and above (P < 0.01). The secretion of both TNF-alpha and IL-6 were only inhibited at the highest dose used (25 microM; P < 0.001). IL-1beta secretion was also inhibited from 12.5 microM onwards (P < 0.01). This inhibition was demonstrated to be caused by down-regulation of inducible enzymes, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), without direct effect upon iNOS or COX-2 enzyme activity. HMP only inhibited iNOS (P < 0.001) and IL-1beta (P < 0.05) gene expression at the highest tested concentration. HMP did not affect the secretion of chemokines IL-8 and monocyte chemotactic protein-1 (MCP-1) and the anti-inflammatory cytokine IL-10. The most striking effect of HMP was its NO inhibitory activity and therefore we conclude that HMP is a selective inhibitor of iNOS.

Curcumin is a highly pleiotropic molecule with significant regulatory effects upon inflammation and inflammatory related diseases. However curcumin has one major important limitation in which it has poor bioavailability. Design of synthetic structural derivatives of curcumin is but one approach that has been used to overcome its poor bioavailability while retaining, or further enhancing, its drug-like effects. We have synthesized a series of curcumin analogues and describe the effects of 2,6-bis-4-(hydroxyl-3-methoxy-benzylidine)-cyclohexanone or BHMC upon nitric oxide and cytokine synthesis in cellular models of inflammation. BHMC showed a significant dose-response inhibitory action upon the synthesis of NO and we have shown that this effect was due to suppression of both iNOS gene and enzyme expression without any effects upon scavenging of nitrite. We also demonstrated that BHMC has a very minimal effect upon iNOS activity with no effect at all upon the secretion of PGE(2) but has a strong inhibitory effect upon MCP-1 and IL-10 secretion and gene expression. Secretion and gene expression of TNF-alpha and IL-6 were moderately inhibited whereas IL-8 and IL-1beta were not altered. We conclude that BHMC selectively inhibits the synthesis of several inflammatory mediators. BHMC should be considered a promising drug lead for preclinical and further pharmacological studies.

Phosphoinositide 3-kinases (PI3Ks) inhibitors have treatment potential for cancer, diabetes, cardiovascular disease, chronic inflammation and asthma. A consensus model consisting of three base classifiers (AODE, kNN, and SVM) trained with 1,283 positive compounds (PI3K inhibitors), 16 negative compounds (PI3K non-inhibitors) and 64,078 generated putative negatives was developed for predicting compounds with PI3K inhibitory activity of IC(50) < or = 10 microM. The consensus model has an estimated false positive rate of 0.75%. Nine novel potential inhibitors were identified using the consensus model and several of these contain structural features that are consistent with those found to be important for PI3K inhibitory activities. An advantage of the current model is that it does not require knowledge of 3D structural information of the various PI3K isoforms, which is not readily available for all isoforms.

Lymphocyte-specific protein tyrosine kinase (Lck) inhibitors have treatment potential for autoimmune diseases and transplant rejection. A support vector machine (SVM) model trained with 820 positive compounds (Lck inhibitors) and 70 negative compounds (Lck noninhibitors) combined with 65 142 generated putative negatives was developed for predicting compounds with a Lck inhibitory activity of IC(50) < or = 10 microM. The SVM model, with an estimated sensitivity of greater than 83% and specificity of greater than 99%, was used to screen 168 014 compounds in the MDDR and was found to have a yield of 45.8% and a false positive rate of 0.52%. The model was also able to identify novel Lck inhibitors and distinguish inhibitors from structurally similar noninhibitors at a false positive rate of 0.27%. To the best of our knowledge, the SVM model developed in this work is the first model with a broad applicability domain and low false positive rate, which makes it very suitable for the virtual screening of chemical libraries for Lck inhibitors.

Prediction and elucidation of pharmacogenetic effects is important for facilitating the development of personalized medicines. Knowledge of polymorphism-induced and other types of drug-response variations is needed for facilitating such studies. Although databases of pharmacogenetic knowledge, polymorphism and toxicogenomic information have appeared, some of the relevant data are provided in separate web-pages and in terms of relatively long descriptions quoted from literatures. To facilitate easy and quick assessment of the relevant information, it is helpful to develop databases that provide all of the information related to a pharmacogenetic effect in the same web-page and in brief descriptions. We developed a database, Pharmacogenetic Effect Database (PharmGED), for providing sequence, function, polymorphism, affected drugs and pharmacogenetic effects. PharmGED can be accessed at http://bidd.cz3.nus.edu.sg/phg/ free of charge for academic use. It currently contains 1825 entries covering 108 disease conditions, 266 distinct proteins, 693 polymorphisms, 414 drugs/ligands cited from 856 references.