Simple and Sensitive Analysis of Blonanserin and Blonanserin C in Human Plasma by Liquid Chromatography Tandem Mass Spectrometry and Its Application

A highly sensitive, simple, and rapid liquid chromatography tandem mass spectrometry method to simultaneously determine blonanserin and blonanserin C in human plasma with AD-5332 as internal standard (IS) was established. A simple direct protein precipitation method was used for the sample pretreatment, and chromatographic separation was performed on a Waters XBridge C(8) (4.6 × 150 mm, 3.5 μm) column. The mobile phase consists of a mixture of 10 mM ammonium formate and 0.1% formic acid in water (A) and 0.1% formic acid in methanol (B). To quantify blonanserin, blonanserin C, and IS, multiple reaction monitoring (MRM) was performed in positive ESI mode. The calibration curve was linear in the concentration range of 0.012–5.78 ng·mL(−1) for blonanserin and 0.023–11.57 ng·mL(−1) for blonanserin C (r (2) > 0.9990). The intra- and interday precision of three quality control (QC) levels in plasma were less than 7.5%. Finally, the current simple, sensitive, and accurate LC-MS/MS method was successfully applied to investigate the pharmacokinetics of blonanserin and blonanserin C in healthy Chinese volunteers

Sugar-Protein Connectivity Impacts on the Immunogenicity of Site-Selective Salmonella O-Antigen Glycoconjugate Vaccines

A series of glycoconjugates with defined connectivity were synthesized to investigate the impact of coupling Salmonella typhimurium O-antigen to different amino acids of CRM197 protein carrier. In particular, two novel methods for site-selective glycan conjugation were developed to obtain conjugates with single attachment site on the protein, based on chemical modification of a disulfide bond and pH-controlled transglutaminase-catalyzed modification of lysine, respectively. Importantly, conjugation at the C186-201 bond resulted in significantly higher anti O-antigen bactericidal antibody titers than coupling to K37/39, and in comparable titers to conjugates bearing a larger number of saccharides. This study demonstrates that the conjugation site plays a role in determining the immunogenicity in mice and one single attachment point may be sufficient to induce high levels of bactericidal antibodies

The Histone H3K79 Methyltransferase Dot1L Is Essential for Mammalian Development and Heterochromatin Structure

Dot1 is an evolutionarily conserved histone methyltransferase specific for lysine 79 of histone H3 (H3K79). In Saccharomyces cerevisiae, Dot1-mediated H3K79 methylation is associated with telomere silencing, meiotic checkpoint control, and DNA damage response. The biological function of H3K79 methylation in mammals, however, remains poorly understood. Using gene targeting, we generated mice deficient for Dot1L, the murine Dot1 homologue. Dot1L-deficient embryos show multiple developmental abnormalities, including growth impairment, angiogenesis defects in the yolk sac, and cardiac dilation, and die between 9.5 and 10.5 days post coitum. To gain insights into the cellular function of Dot1L, we derived embryonic stem (ES) cells from Dot1L mutant blastocysts. Dot1L-deficient ES cells show global loss of H3K79 methylation as well as reduced levels of heterochromatic marks (H3K9 di-methylation and H4K20 tri-methylation) at centromeres and telomeres. These changes are accompanied by aneuploidy, telomere elongation, and proliferation defects. Taken together, these results indicate that Dot1L and H3K79 methylation play important roles in heterochromatin formation and in embryonic development

Responses of lakeshore herbaceous plant guilds to altered water level fluctuations in Yangtze floodplain lakes, China

River–lake disconnection has led to alterations in natural water level fluctuations (WLFs) in lakes along the middle and lower reaches of the Yangtze River to various extents. To predict the effects of altered WLFs on lakeshore vegetation, lakeshore herbaceous plant guilds (LPGs) in six Yangtze floodplain lakes were classified in this study. The lakeshore herbaceous plant distribution pattern, main influencing factors, and responses of different LPGs to altered WLFs were analyzed. A total of 130 plant species were identified from the six lakes. With the increase in the amplitude of WLFs, the total species number in the six lakes showed a hump–shaped trend, whereas the lakeshore herbaceous plant coverage showed a positive correlation. The Qili Lake with the largest WLFs had the lowest species number and highest plant coverage. The species numbers, average biomass, and coverage of lakeshore herbaceous plants in all the six lakes were obviously higher in spring than in autumn. The lakeshore herbaceous plants in the six lakes were classified into eight guilds. Small differences in pH, organic matter, total nitrogen, and total phosphorus were detected in the substrate among the eight LPGs, but obvious differences in soil moisture content and duration and depth of submergence were observed. The canonical correspondence analysis showed that the main environmental factors affecting the distribution of different LPGs in the six lakes in spring and autumn were the amplitude of WLFs and duration of submergence. The regression analyses showed that the biomass of perennial mesophyte with erect stem guild and perennial hygrophyte with well–developed rhizome guild responded sensitively to alterations in the amplitude of WLFs and duration of submergence, and the guilds could be considered as indicator groups at a regional scale. Morphological parameters such as plant height, stem diameter, leaf thickness, and specific leaf area of the eight LPGs showed no significant linear relationships with the increase in WLFs. The use of these parameters in the evaluations of alterations in WLFs needs to be further analyzed

Simultaneous Kinetic Characterization of Multiple Protein Forms by Top Down Mass Spectrometry

Top down mass spectrometry, using a Fourier transform instrument, has unique capabilities for biomolecule kinetic studies, in that the concentration of large molecules in a reaction mixture can be monitored simultaneously from its mass spectrum produced by electrospray ionization. This is demonstrated with enzyme modifications occurring in the biosynthesis of the thiazole moiety of thiamin phosphate. The formation rate of ThiS-thiocarboxylate from ThiS was determined from the relative abundance of the corresponding m/z 10162 and 10146 isotopic peak clusters for all the observable charge states in the mass spectra measured at different reaction times. Even without measuring standard ionization efficiencies, the rate and precision of 0.018 ± 0.004 min−1 agree well with the 0.027 ± 0.003 min−1 obtained with a radiochemical assay, which requires a separate derivatization step. To illustrate the simultaneous characterization of the reaction kinetics of a native enzyme and its mutant, the imine formation rate of ThiG and its substrate DXP was compared between the native protein (Mr = 26803.9) and its E98A (Mr = 26745.9) or D182A (Mr = 26759.9) mutant in the same reaction mixture. The kinetic data show clearly that neither the E98 nor the D182 residues participate in the imine formation. The high resolution and MS/MS capabilities of FTMS should make possible the extension of this kinetics approach to far more complicated systems, such as simultaneous monitoring of 24 native, intermediate, and reduced forms in the reductive unfolding of a mixture of ribonuclease A and the five isoforms of ribonuclease B. Stable intermediates with different SS bonding (same molecular weight) can be differentiated by MS/MS, while molecular ions differing by only 2 Da are distinguished clearly by synthesizing isotopically depleted proteins

a systematic substrate screen links CDK4/6 to senescence suppression through FOXM1

Cyclin D-CDKs (CDK4, CDK6) are critical regulators of cell cycle entry, and their aberrant expression and activation is observed in the majority of human cancers. However, it is currently not completely understood by which cellular mechanisms CDK4/6 promote tumorigenesis, largely due to the limited number of identified substrates. Here we applied a proteome-wide substrate screen and demonstrate that individual cyclin D-CDK complexes possess major differences in substrate specificity. We identified the Forkhead Box M1 (FOXM1) transcription factor as a functionally critical phosphorylation target. While modification of its N-terminal sequence is linked to stabilization of the protein by preventing CDH1/APC-mediated degradation, phosphorylation of the transactivation domain leads to direct transcriptional activation, resulting in suppression of cellular senescence. Moreover, FOXM1 is required for CDK4-induced expression of proliferation-associated genes, including cyclin E. Accordingly, acute inhibition of CDK4/6 activity in cancer cells disables FOXM1, results in downregulation of cell cycle and DNA repair genes, and enforces a ROS-dependent senescence program. Thus, pharmacological inhibition of CDK4/6 catalytic activity might be particularly effective in tumors that highly depend on FOXM1

Simple and Sensitive Analysis of Blonanserin and Blonanserin C in Human Plasma by Liquid Chromatography Tandem Mass Spectrometry and Its Application

A highly sensitive, simple, and rapid liquid chromatography tandem mass spectrometry method to simultaneously determine blonanserin and blonanserin C in human plasma with AD-5332 as internal standard (IS) was established. A simple direct protein precipitation method was used for the sample pretreatment, and chromatographic separation was performed on a Waters XBridge C8 (4.6×150 mm, 3.5 μm) column. The mobile phase consists of a mixture of 10 mM ammonium formate and 0.1% formic acid in water (A) and 0.1% formic acid in methanol (B). To quantify blonanserin, blonanserin C, and IS, multiple reaction monitoring (MRM) was performed in positive ESI mode. The calibration curve was linear in the concentration range of 0.012–5.78 ng·mL−1 for blonanserin and 0.023–11.57 ng·mL−1 for blonanserin C (r2>0.9990). The intra- and interday precision of three quality control (QC) levels in plasma were less than 7.5%. Finally, the current simple, sensitive, and accurate LC-MS/MS method was successfully applied to investigate the pharmacokinetics of blonanserin and blonanserin C in healthy Chinese volunteers