CDK5-dependent inhibitory phosphorylation of Drp1 during neuronal maturation

Mitochondrial functions are essential for the survival and function of neurons. Recently, it has been demonstrated that mitochondrial functions are highly associated with mitochondrial morphology, which is dynamically changed by the balance between fusion and fission. Mitochondrial morphology is primarily controlled by the activation of dynamin-related proteins including dynamin-related protein 1 (Drp1), which promotes mitochondrial fission. Drp1 activity is regulated by several post-translational modifications, thereby modifying mitochondrial morphology. Here, we found that phosphorylation of Drp1 at serine 616 (S616) is mediated by cyclin-dependent kinase 5 (CDK5) in post-mitotic rat neurons. Perturbation of CDK5 activity modified the level of Drp1^S616 phosphorylation and mitochondrial morphology in neurons. In addition, phosphorylated Drp1^S616 preferentially localized as a cytosolic monomer compared with total Drp1. Furthermore, roscovitine, a chemical inhibitor of CDKs, increased oligomerization and mitochondrial translocation of Drp1, suggesting that CDK5-dependent phosphorylation of Drp1 serves to reduce Drp1''s fission-promoting activity. Taken together, we propose that CDK5 has a significant role in the regulation of mitochondrial morphology via inhibitory phosphorylation of Drp1^S616 in post-mitotic neurons.     

Optimized precursor ion selection for labile ions in a linear ion trap mass spectrometer and its impact on quantification using selected reaction monitoring

The fragmentation of fragile ions during the application of an isolation waveform for precursor ion selection and the resulting loss of isolated ion intensity is well‐known in ion trap mass spectrometry (ITMS). To obtain adequate ion intensity in the selected reaction monitoring (SRM) of fragile precursor ions, a wider ion isolation width is required. However, the increased isolation width significantly diminishes the selectivity of the channels chosen for SRM, which is a serious problem for samples with complex matrices. The sensitive and selective quantification of many lipid molecules, including ceramides from real biological samples, using a linear ion trap mass spectrometer is also hindered by the same problem because of the ease of water loss from protonated ceramide ions. In this study, a method for the reliable quantification of ceramides using SRM with near unity precursor ion isolation has been developed for ITMS by utilizing alternative precursor ions generated by in‐source dissociation. The selected precursor ions allow the isolation of ions with unit mass width and the selective analysis of ceramides using SRM with negligible loss of sensitivity. The quantification of C18:0‐, C24:0‐ and C24:1‐ceramides using the present method shows excellent linearity over the concentration ranges from 6 to 100, 25 to 1000 and 25 to 1000 nM, respectively. The limits of detection of C18:0‐, C24:0‐ and C24:1‐ceramides were 0.25, 0.25 and 5 fmol, respectively. The developed method was successfully applied to quantify ceramides in fetal bovine serum. Copyright © 2014 John Wiley & Sons, Ltd.     

Inhibition of phospholipase D2 induces autophagy in colorectal cancer cells

Autophagy is a conserved lysosomal self-digestion process used for the breakdown of long-lived proteins and damaged organelles, and it is associated with a number of pathological processes, including cancer. Phospholipase D (PLD) isozymes are dysregulated in various cancers. Recently, we reported that PLD1 is a new regulator of autophagy and is a potential target for cancer therapy. Here, we investigated whether PLD2 is involved in the regulation of autophagy. A PLD2-specific inhibitor and siRNA directed against PLD2 were used to treat HT29 and HCT116 colorectal cancer cells, and both inhibition and genetic knockdown of PLD2 in these cells significantly induced autophagy, as demonstrated by the visualization of light chain 3 (LC3) puncta and autophagic vacuoles as well as by determining the LC3-II protein level. Furthermore, PLD2 inhibition promoted autophagic flux via the canonical Atg5-, Atg7- and AMPK-Ulk1-mediated pathways. Taken together, these results suggest that PLD2 might have a role in autophagy and that its inhibition might provide a new therapeutic basis for targeting autophagy.     

Pd‐Catalyzed Cycloisomerization of 4‐Aza‐1,6‐enynes to 3‐Aza‐bicyclo[4.1.0]hept‐2‐enes

A method for the synthesis of bicyclo[4.1.0]heptenes from 1,6‐enynes through Pd‐catalyzed cycloisomerization has been developed. N‐ and O‐tethered 1,6‐enynes were successfully transformed to their corresponding 3‐aza‐ and 3‐oxabicyclo[4.1.0]heptenes in reasonable‐to‐high yields using the catalysts [PdCl₂(CH₃CN)₂]/P(OPh)₃ or [Pd(maleimidate)₂(PPh₃)₂] in toluene. The computational calculations using density functional theory indicate that [PdCl₂{P(OPh)₃}] in the oxidation state Pd^II acts as the active catalyst species for the formation of 3‐azabicyclo[4.1.0]heptenes through 6‐endo‐dig cyclization.     

Synergism of cellulosic nanowhiskers and graft structure in stereocomplex-based materials: formation in solution and a stereocomplex memory study

Stereocomplexation is one of the approaches to improve polylactide's properties. Along with improving its properties, it also limits stereocomplex formation through solution and stereocomplex memory. The graft structure and presence of nanoparticles have a synergetic effect, improving the stereocomplex formation and its memory. The bio-stereocomplex-nanocomposite materials are generated by stereocomplexation of polylactide-graft-acetylated cellulosic nanowhiskers in the solution. The graft structure containing well-distributed acetylated cellulose nanowhiskers results in unusual stereocomplexation in the solution and influences the stereocomplex memory of the bio-stereocomplex-nanocomposite materials. Perfect stereocomplexes are easily obtained in a relatively short mixing time (5 min) from various solution concentrations up to 20 % (w/v). The bio-stereocomplex-nanocomposites have excellent stereocomplex memory to re-form the stereocomplex after melting, which is the main limitation of stereocomplex materials in industrial processes. This fully bio-based material is a potential ecofriendly candidate for the future.     

Phosphonated Near‐Infrared Fluorophores for Biomedical Imaging of Bone

The conventional method for creating targeted contrast agents is to conjugate separate targeting and fluorophore domains. A new strategy is based on the incorporation of targeting moieties into the non‐delocalized structure of pentamethine and heptamethine indocyanines. Using the known affinity of phosphonates for bone minerals in a model system, two families of bifunctional molecules that target bone without requiring a traditional bisphosphonate are synthesized. With peak fluorescence emissions at approximately 700 or 800 nm, these molecules can be used for fluorescence‐assisted resection and exploration (FLARE) dual‐channel imaging. Longitudinal FLARE studies in mice demonstrate that phosphonated near‐infrared fluorophores remain stable in bone for over five weeks, and histological analysis confirms their incorporation into the bone matrix. Taken together, a new strategy for creating ultra‐compact, targeted near‐infrared fluorophores for various bioimaging applications is described.     

Hierarchical Self‐Assembly of Poly‐Pseudorotaxanes into Artificial Microtubules

Hierarchical self‐assembly of building blocks over multiple length scales is ubiquitous in living organisms. Microtubules are one of the principal cellular components formed by hierarchical self‐assembly of nanometer‐sized tubulin heterodimers into protofilaments, which then associate to form micron‐length‐scale, multi‐stranded tubes. This peculiar biological process is now mimicked with a fully synthetic molecule, which forms a 1:1 host‐guest complex with cucurbit[7]uril as a globular building block, and then polymerizes into linear poly‐pseudorotaxanes that associate laterally with each other in a self‐shape‐complementary manner to form a tubular structure with a length over tens of micrometers. Molecular dynamic simulations suggest that the tubular assembly consists of eight poly‐pseudorotaxanes that wind together to form a 4.5 nm wide multi‐stranded tubule.     

Simultaneous separation and determination of 20 potential adulterant antigout and antiosteoporosis pharmaceutical compounds in herbal food products using LC with electrospray ionization MS/MS and LC with quadrupole‐time‐of‐flight MS

An analytical method for the simultaneous and reliable determination of 20 antigout and antiosteoporosis pharmaceutical compounds in adulterated health food products was developed using liquid chromatography with electrospray ionization tandem mass spectrometry and liquid chromatography with quadrupole‐time‐of‐flight mass spectrometry. The method was validated through the determination of specificity, linearity, limit of detection, and limit of quantification, method detection limit, method quantitation limit, precision, accuracy, recovery, and stability. The matrix effect was also determined. The validation results of the developed method are as follows: for solid and liquid blank samples, limits of detection ranged from 0.05 to 5.00 ng/mL and limits of quantification ranged from 0.15 to 15.00 ng/mL. Linearity was acceptable, and the correlation coefficients (R²) were ≥0.99 for all target compounds. Both intra and interday precision were less than 9.16% RSD, and accuracies ranged from 95.31 to 116.68%. Mean recoveries for different types of dietary supplements classified as powders, liquids, tablets, and capsules were found to be 80.81 to 117.62% with less than 15.00% relative standard deviation. The stability of the standard mixture solution was less than 11.72% relative standard deviation after 48 h. By the proposed method, the presence of dexamethasone was determined in seized herbal food products at concentrations that ranged from 126 to 215 µg/g.