Inhibition mechanism of testis-expressed gene 14 (TEX14) in cytokinetic abscission: Well-tempered metadynamics simulation studies.

Cytokinesis requires a apoptosis-linked gene 2 interacting protein X (ALIX) and a 55 kDa midbody centrosomal protein (CEP55) to activate the cell abscission in somatic cells. However, in germ cells, CEP55 forms intercellular bridges with testis-expressed gene 14 (TEX14), which blocks the cell abscission. These intercellular bridges play important roles in the synchronization of the germ cells and facilitate the coordinated passage of organelles and molecules between germ cells. If TEX14 is intentionally removed, intercellular bridges are disrupted, leading to sterility. Hence, a deeper understanding regarding the roles of TEX14 can provide significant insights into the inactivation of abscission and the inhibition of proliferation in cancer cells. Previous experimental studies have shown that the high affinity and low dissociation rate of TEX14 for CEP55 prevent ALIX from binding CEP55 and inactivate the germ cell abscission. However, detailed information about how TEX14 interacts with CEP55 to prevent the cell abscission is still lacking. To gain more specific insights into the interactions between CEP55 and TEX14 and the difference in reactivity between TEX14 and ALIX, we performed well-tempered metadynamics simulations of these protein complexes using atomistic models of CEP55, TEX14, and ALIX. We identified the major binding residues of TEX14 and ALIX with CEP55 by using 2D Gibbs free energy evaluations, the results of which are consistent with previous experimental studies. Our results may help design synthetic TEX14 mimicking peptides, which can bind CEP55 and facilitate the inactivation of abscission in abnormal cells, including cancer cells.

A motor neuron strategy to save time and energy in neurodegeneration: adaptive protein stoichiometry.

Neurofilament proteins (Nf) are a biomarker of disease progression in amyotrophic lateral sclerosis (ALS). This study investigated whether there are major differences in expression from in vivo measurements of neurofilament isoforms, from the light chain, NfL (68 kDa), compared with larger proteins, the medium chain (NfM, 150 kDa) and the heavy (NfH, 200-210 kDa) chains in ALS patients and healthy controls. New immunological methods were combined with Nf subunit stoichiometry calculations and Monte Carlo simulations of a coarse-grained Nf brush model. Based on a physiological Nf subunit stoichiometry of 7 : 3 : 2 (NfL:NfM:NfH), we found an 'adaptive' Nf subunit stoichiometry of 24 : 2.4 : 1.6 in ALS. Adaptive Nf stoichiometry preserved NfL gyration radius in the Nf brush model. The energy and time requirements for Nf translation were 56 ± 27k ATP (5.6 h) in control subjects compared to 123 ± 102k (12.3 h) in ALS with 'adaptive' (24:2.4:1.6) Nf stoichiometry (not significant) and increased significantly to 355 ± 330k (35.5 h) with 'luxury' (7:3:2) Nf subunit stoichiometry (p < 0.0001 for each comparison). Longitudinal disease progression-related energy consumption was highest with a 'luxury' (7:3:2) Nf stoichiometry. Therefore, an energy and time-saving option for motor neurons is to shift protein expression from larger to smaller (cheaper) subunits, at little or no costs on a protein structural level, to compensate for increased energy demands.

Developments in FT-ICR MS instrumentation, ionization techniques, and data interpretation methods for petroleomics.

Because of the increasing importance of heavy and unconventional crude oil as an energy source, there is a growing need for petroleomics: the pursuit of more complete and detailed knowledge of the chemical compositions of crude oil. Crude oil has an extremely complex nature; hence, techniques with ultra-high resolving capabilities, such as Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS), are necessary. FT-ICR MS has been successfully applied to the study of heavy and unconventional crude oils such as bitumen and shale oil. However, the analysis of crude oil with FT-ICR MS is not trivial, and it has pushed analysis to the limits of instrumental and methodological capabilities. For example, high-resolution mass spectra of crude oils may contain over 100,000 peaks that require interpretation. To visualize large data sets more effectively, data processing methods such as Kendrick mass defect analysis and statistical analyses have been developed. The successful application of FT-ICR MS to the study of crude oil has been critically dependent on key developments in FT-ICR MS instrumentation and data processing methods. This review offers an introduction to the basic principles, FT-ICR MS instrumentation development, ionization techniques, and data interpretation methods for petroleomics and is intended for readers having no prior experience in this field of study.

Elucidating molecular structures of nonalkylated and short-chain alkyl (n < 5, (CH2)n) aromatic compounds in crude oils by a combination of ion mobility and ultrahigh-resolution mass spectrometries and theoretical collisional cross-section calculations.

Ultrahigh-resolution mass spectrometry has allowed the determination of elemental formulas of the compounds comprising crude oils. However, elucidating molecular structures remains an analytical challenge. Herein, we propose and demonstrate an approach combining ion mobility mass spectrometry (IM-MS), ultrahigh-resolution mass spectrometry, and theoretical collisional cross-section (CCS) calculations to determine the molecular structures of aromatic compounds found in crude oils. The approach is composed of three steps. First, chemical structures are suggested based on the elemental formulas determined from ultrahigh-resolution mass spectra. Second, theoretical CCS values are calculated based on these proposed structures. Third, the calculated CCS values of the proposed structures are compared with experimentally determined CCS values from IM-MS data to provide proposed structures. For proof of concept, 31 nonalkylated and short-chain alkyl (n < 5, (CH2)n) aromatic compounds commonly observed in crude oils were analyzed. Theoretical and experimental CCS values matched within a 5% RMS error. This approach was then used to propose structures of compounds in selected m/z regions of crude oil samples. Overall, the combination of ion mobility mass spectrometry, ultrahigh-resolution mass spectrometry, and theoretical calculations was shown to be a useful tool for elucidating chemical structures of compounds in complex mixtures.

Application of atmospheric pressure photo ionization hydrogen/deuterium exchange high-resolution mass spectrometry for the molecular level speciation of nitrogen compounds in heavy crude oils.

We report here for the first time the application of atmospheric pressure photo ionization hydrogen/deuterium exchange (APPI HDX) coupled to high-resolution mass spectrometry for molecular level speciation of nitrogen containing compounds in crude oils. The speciation was done based on different combinations of ions produced from nitrogen containing compounds with various functional groups. To prove the concept, 20 nitrogen containing standard compounds were analyzed. As a result, it was shown that the nitrogen containing compound (M) with a primary amine functional group mainly produced a combination of [M - 2H + 2D](•+) and ([M - 2H + 2D] + D)(+) ions, one with a secondary amine including alkylated or phenylated pyrrole a combination of [M - H + D](•+) and ([M - H + D] + D)(+), one with a tertiary amine including N-alkylated or phenylated pyrrole a combination of [M](•+) and [M + D](+), and one with a pyridine functional group mostly [M + D](+) ions. The concept was successfully applied to do nitrogen speciation of resins fractions of two oil samples. Combined with the subsequent investigation of double bond equivalence distribution, it was shown that resins of Qinhuangdao crude oil sample contained mostly alkylated pyrrole and N-alkylated pyrrole type compounds but resins of shale oil extract contained mostly pyridine type nitrogen compounds. It was also shown that the speciation of individual elemental composition was also possible by use of this method. Overall, this study clearly shows that atmospheric pressure photo ionization hydrogen/deuterium exchange (APPI HDX) coupled to high-resolution mass spectrometry is a powerful analytical method to do nitrogen speciation of crude oil compounds at the molecular level.

Characterization of crude oils at the molecular level by use of laser desorption ionization Fourier-transform ion cyclotron resonance mass spectrometry.

In this study, laser desorption ionization (LDI) coupled to Fourier-transform ion cyclotron resonance mass spectrometry (FTICR MS) was applied to study crude oils at the molecular level. Molecular ions were the major type of ion detected by (+) mode, and deprotonated and radical anions were the major ions observed by (-) mode LDI FTICR MS. N(1) and hydrocarbon classes were dominant in the class distribution plots obtained by (+) LDI FTICR MS, but other heteroatom classes, including O(x) and S(1), were abundant in plots obtained by (-) LDI FTICR MS. Detailed analysis of double-bond equivalence (DBE) vs carbon number plots revealed that LDI FTICR MS is more sensitive toward polyaromatic compounds than mono- or dicyclic-aromatic compounds. However, nonaromatic and aromatic O(2) compounds could be detected simultaneously. An abundance of nonaromatic O(2) compounds (presumably naphthenic acids) are correlated with total acid numbers, but O(2) compounds with condensed structures are not. Overall, this study shows that LDI FTICR MS can be successfully used to study crude oils at the molecular level.

Planar limit-assisted structural interpretation of saturates/aromatics/resins/asphaltenes fractionated crude oil compounds observed by Fourier transform ion cyclotron resonance mass spectrometry.

Planar limits, defined as lines generated by connecting maximum double-bond equivalence (DBE) values at given carbon numbers, are proposed as a means of predicting and understanding the molecular structure of compounds in crude oil. The slopes and y-intercepts of the lines are determined by the DBE/carbon number ratios of functional groups defining the planar limits. For example, the planar limit generated by a serial addition of saturated cyclic rings has a slope of 0.25. The planar limit formed by the linear and nonlinear addition of benzene rings yields lines with slopes of 0.75 and 1, respectively. The y-intercepts of these lines were determined by additional functional groups added within a series of molecules. Plots of DBE versus carbon number for S(1) class compounds observed by Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) showed that saturates/aromatics/resins/asphaltenes (SARA) fractions exhibited unique slopes and y-intercepts. The slope of the planar limit observed from a saturates fraction matched well with the slope of a planar limit generated by the serial addition of saturated cyclic rings. The slopes of planar limits of aromatics and resins fractions were very similar to that obtained from the linear addition of benzene rings. Finally, the slope of the asphaltenes fraction was almost identical to the slope obtained from the nonlinear addition of benzene rings. Simulated and experimental data show that SARA fractions exhibit different molecular structure characteristics. On the basis of the slope and y-intercept of the planar limit, the structures of molecules in SARA fractions were predicted and suggested. The use of planar limits for structural interpretation is not limited to crude oil compounds but can also be used to study other organic mixtures such as humic substances or metabolites.

Antiobesity Effect of Prebiotic Polyphenol-Rich Grape Seed Flour Supplemented with Probiotic Kefir-Derived Lactic Acid Bacteria.

The interaction between prebiotics and probiotics may exert synergistic health benefits. This study investigated the combined effects of polyphenol-rich wine grape seed flour (GSF), a prebiotic, and lactic acid bacteria (LAB) derived from kefir, a probiotic, on obesity-related metabolic disease in high-fat diet (HFD) induced obese (DIO) mice. DIO mice were fed with HFD with 6% microcrystalline cellulose (CON) or HFD supplemented with GSF (5% or 10% GSF), HFD with LAB orally administrated (LAB), or HFD with a combination of GSF and LAB orally administrated (GSF+LAB) for 9 weeks. The vehicle, saline, was also orally administered to the CON and GSF groups. In comparison to CON, all GSF and LAB groups showed a reduction ( P < 0.05) in HF-induced weight gain, liver and adipose tissue weights, plasma lipid concentrations, insulin resistance, and glucose intolerance. The combination of 10% GSF and LAB showed synergistic effects ( P < 0.05) on body weight gain, plasma insulin and total cholesterol concentrations, and cecum propionate contents. Plasma zonulin and cecum propionate concentrations and intestinal FXR gene expression were ( P < 0.05) correlated with body weight gain. A pathway analysis of microarray data of adipose tissue showed that the combination of GSF and LAB affected genes involved in metabolic and immunological diseases, including inflammasome complex assembly ( P < 0.05). In conclusion, a combination of GSF and LAB inhibited HF-induced obesity and inflammation via alterations in intestinal permeability and adipocyte gene expression.

Ternary Organic Photovoltaics Prepared by Sequential Deposition of Single Donor and Binary Acceptors.

Binary organic photovoltaics (OPVs) fabricated by single-step (SS) deposition of a binary blend of polymer (or small molecule) donor and fullerene acceptor (SS binary OPV) are widely utilized. To improve the OPV performance, SS ternary OPVs utilizing a ternary blend consisting of two (or one) electron donor(s) and one (or two) electron acceptor(s) have been studied. SS ternary OPVs require more sensitive and complex optimization processes to optimize bulk heterojunctions with bicontinuous nanoscale phase separation of the donor and acceptor. We demonstrated a novel ternary OPV fabricated by sequential (SQ) deposition of a single polymer donor and a binary mixture consisting of a phenyl-C71-butyric acid methyl ester (PCBM) and nonfullerene acceptor, 3,9-bis(2-methylene-(3-(1,1-dicyanomethylene)-indanone))-5,5,11,11-tetrakis(4-hexylphenyl)-dithieno[2,3- d:2,3'- d']- s-indaceno[1,2- b:5,6- b']dithiophene (ITIC). In the SQ ternary OPV, PCBM effectively created a bicontinuous pathway for charge transport with a polymer, and ITIC mainly enhanced light absorption and photovoltage. This complementary effect was not observed in an SS ternary OPV utilizing the same donor and acceptors. Due to these complementary effects, the SQ ternary OPV exhibited a power conversion efficiency of 6.22%, which was 52 and 37% higher than that of the SQ binary OPV and the SS ternary OPV, respectively. In addition, the thermal stability of the SQ ternary OPV was found to be superior to that of the SS ternary OPV.

Distinct Fragmentation Pathways of Anticancer Drugs Induced by Charge-Carrying Cations in the Gas Phase.

With the growth of the pharmaceutical industry, structural elucidation of drugs and derivatives using tandem mass spectrometry (MS2) has become essential for drug development and pharmacokinetics studies because of its high sensitivity and low sample requirement. Thus, research seeking to understand fundamental relationships between fragmentation patterns and precursor ion structures in the gas phase has gained attention. In this study, we investigate the fragmentation of the widely used anticancer drugs, doxorubicin (DOX), vinblastine (VBL), and vinorelbine (VRL), complexed by a singly charged proton or alkali metal ion (Li+, Na+, K+) in the gas phase. The drug-cation complexes exhibit distinct fragmentation patterns in tandem mass spectra as a function of cation size. The trends in fragmentation patterns are explicable in terms of structures derived from ion mobility mass spectrometry (IM-MS) and theoretical calculations. Graphical Abstract ᅟ.

Supercritical fluid chromatography coupled with in-source atmospheric pressure ionization hydrogen/deuterium exchange mass spectrometry for compound speciation.

An experimental setup for the speciation of compounds by hydrogen/deuterium exchange (HDX) with atmospheric pressure ionization while performing chromatographic separation is presented. The proposed experimental setup combines the high performance supercritical fluid chromatography (SFC) system that can be readily used as an inlet for mass spectrometry (MS) and atmospheric pressure photo ionization (APPI) or atmospheric pressure chemical ionization (APCI) HDX. This combination overcomes the limitation of an approach using conventional liquid chromatography (LC) by minimizing the amount of deuterium solvents used for separation. In the SFC separation, supercritical CO2 was used as a major component of the mobile phase, and methanol was used as a minor co-solvent. By using deuterated methanol (CH3OD), AP HDX was achieved during SFC separation. To prove the concept, thirty one nitrogen- and/or oxygen-containing standard compounds were analyzed by SFC-AP HDX MS. The compounds were successfully speciated from the obtained SFC-MS spectra. The exchange ions were observed with as low as 1% of CH3OD in the mobile phase, and separation could be performed within approximately 20min using approximately 0.24 mL of CH3OD. The results showed that SFC separation and APPI/APCI HDX could be successfully performed using the suggested method.

Structural Characterization of Anticancer Drug Paclitaxel and Its Metabolites Using Ion Mobility Mass Spectrometry and Tandem Mass Spectrometry.

Paclitaxel (PTX) is a popular anticancer drug used in the treatment of various types of cancers. PTX is metabolized in the human liver by cytochrome P450 to two structural isomers, 3'-p-hydroxypaclitaxel (3p-OHP) and 6α-hydroxypaclitaxel (6α-OHP). Analyzing PTX and its two metabolites, 3p-OHP and 6α-OHP, is crucial for understanding general pharmacokinetics, drug activity, and drug resistance. In this study, electrospray ionization ion mobility mass spectrometry (ESI-IM-MS) and collision induced dissociation (CID) are utilized for the identification and characterization of PTX and its metabolites. Ion mobility distributions of 3p-OHP and 6α-OHP indicate that hydroxylation of PTX at different sites yields distinct gas phase structures. Addition of monovalent alkali metal and silver metal cations enhances the distinct dissociation patterns of these structural isomers. The differences observed in the CID patterns of metalated PTX and its two metabolites are investigated further by evaluating their gas-phase structures. Density functional theory calculations suggest that the observed structural changes and dissociation pathways are the result of the interactions between the metal cation and the hydroxyl substituents in PTX metabolites.

Optimization and Application of APCI Hydrogen-Deuterium Exchange Mass Spectrometry (HDX MS) for the Speciation of Nitrogen Compounds.

A systematic study was performed to investigate the utility of atmospheric pressure chemical ionization hydrogen-deuterium exchange mass spectrometry (APCI HDX MS) to identify the structures of nitrogen-containing aromatic compounds. First, experiments were performed to determine the optimized experimental conditions, with dichloromethane and CH(3)OD found to be good cosolvents for APCI HDX. In addition, a positive correlation between the heated capillary temperature and the observed HDX signal was observed, and it was suggested that the HDX reaction occurred when molecules were contained in the solvent cluster. Second, 20 standard nitrogen-containing compounds were analyzed to investigate whether speciation could be determined based on the different types of ions produced from nitrogen-containing compounds with various functional groups. The number of exchanges occurring within the compounds correlated well with the number of active hydrogen atoms attached to nitrogen, and it was confirmed that APCI HDX MS could be used to determine speciation. The results obtained by APCI HDX MS were combined with the subsequent investigation of the double bond equivalence distribution and indicated that resins of shale oil extract contained mostly pyridine type nitrogen compounds. This study confirmed that APCI HDX MS can be added to previously reported chemical ionization, electrospray ionization, and atmospheric pressure photo ionization-based HDX methods, which can be used for structural elucidation by mass spectrometry.

Application of phase correction to improve the interpretation of crude oil spectra obtained using 7 T Fourier transform ion cyclotron resonance mass spectrometry.

In this study, a phase-correction technique was applied to the study of crude oil spectra obtained using a 7 T Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). 7 T FT-ICR MS had not been widely used for oil analysis due to the lower resolving power compared with high field FT-ICR MS. For low field instruments, usage of data that has not been phase-corrected results in an inability to resolve critical mass splits of C3 and SH4 (3.4 mDa), and (13)C and CH (4.5 mDa). This results in incorrect assignments of molecular formulae, and discontinuous double bond equivalents (DBE) and carbon number distributions of S1, S2, and hydrocarbon classes are obtained. Application of phase correction to the same data, however, improves the reliability of assignments and produces continuous DBE and carbon number distributions. Therefore, this study clearly demonstrates that phase correction improves data analysis and the reliability of assignments of molecular formulae in crude oil anlayses.

The comparison of naturally weathered oil and artificially photo-degraded oil at the molecular level by a combination of SARA fractionation and FT-ICR MS.

Two sets of oil samples, one obtained from different weathering stages of the M/V Hebei Spirit oil spill site and the other prepared by an in vitro photo-degradation experiment, were analyzed and compared at the molecular level by atmospheric pressure photo-ionization coupled with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). For a more detailed comparison at the molecular level, the oil samples were separated into saturate, aromatic, resin, and asphaltene (SARA) fractions before MS analysis. Gravimetric analysis of the SARA fractions revealed a decreased weight percentage of the aromatic fraction and an increased resin fraction in both sets of samples. Molecular-level investigations of the SARA fractions showed a significant reduction in the S1 class in the saturate fraction and increase of S1O1 class compounds with high DBE values in resin fraction. Levels of N1 and N1O1 class compounds resulting in protonated ions (presumably basic nitrogen compounds) increased after degradation compared to compounds generating molecular ions (presumably non-basic nitrogen compounds). This study revealed changes occurring in heteroatom polar species of crude oils such as sulfur and nitrogen containing compounds that have not been easily detected with conventional GC based techniques.

Hepatic and splenic infarction and bowel ischemia following endoscopic ultrasound-guided celiac plexus neurolysis.

Endoscopic ultrasound-guided celiac plexus neurolysis (EUS-CPN) is a well-established intervention to palliate malignant pain. We report a patient who developed hepatic and splenic infarction and bowel ischemia following EUS-CPN. A 69-year-old man with known lung cancer and pancreatic metastasis was transferred for debilitating, significant epigastric pain for several months. The patient underwent EUS-CPN to palliate the pain. After the procedure, the patient complained continuously of abdominal pain, nausea, and vomiting; hematemesis and hematochezia were newly developed. Abdominal computed tomography revealed infarction of the liver and spleen and ischemia of the stomach and proximal small bowel. On esophagogastroduodenoscopy, hemorrhagic gastroduodenitis, and multiple gastric ulcers were noted without active bleeding. The patient expired on postoperative day 27 despite the best supportive care.

Culture Conditions for the Mycelial Growth of Ganoderma applanatum.

Ganoderma applanatum is one of the most popular medicinal mushrooms due to the various biologically active components it produces. This study was conducted to obtain basic information regarding the mycelial culture conditions of Ganoderma applanatum. Based on the colony diameter and mycelial density, PDA, YMA and MCM media were suitable for the mycelial growth of the mushroom. The optimum temperature for mycelial growth was found to be 25~30℃. The optimum carbon and nitrogen sources were mannose and dextrin, respectively, and the optimum C/N ratio was 2 to 10 when 2% glucose was used. Other minor components required for the optimal growth included thiamine-HCl and biotin as vitamins, succinic acid and lactic acid as organic acids, and MgSO4·7H2O, KH2PO4 and NaCl as mineral salts.