N-Glycosidase treatment with 18O labeling and de novo sequencing argues for flagellin FliC glycopolymorphism in Pseudomonas aeruginosa

In prokaryote organisms, N-glycosylation of proteins is often correlated to cell–cell recognition and extracellular events. Those glycoproteins are potential targets for infection control. To date, many surface-glycosylated proteins from bacterial pathogens have been described. However, N-linked Pseudomonas surface-associated glycoproteins remain underexplored. We report a combined enrichment and labeling strategy to identify major glycoproteins on the outside of microorganisms. More precisely, bacteria were exposed to a mix of biotinylated lectins able to bind with glycoproteins. The latter were then recovered by avidin beads, digested with trypsin, and submitted to mass spectrometry. The targeted mixture of glycoproteins was additionally deglycosylated in the presence of H₂ ¹⁸O to incorporate ¹⁸O during PNGase F treatment and were also analyzed using mass spectrometry. This approach allowed us to identify a few tens of potential N-glycoproteins, among which flagellin FliC was the most abundant. To detect the possible sites of FliC modifications, a de novo sequencing step was also performed to discriminate between spontaneous deamidation and N-glycan loss. This approach led to the proposal of three potential N-glycosylated sites on the primary sequence of FliC: N26, N69, and N439, with two of these three asparagines belonging to an N-X-(S/T) consensus sequence. These observations suggest that flagellin FliC is a heterogeneous protein mixture containing both O- and N-glycoforms.

PCR-based detection of gene transfer vectors: application to gene doping surveillance

Athletes who illicitly use drugs to enhance their athletic performance are at risk of being banned from sports competitions. Consequently, some athletes may seek new doping methods that they expect to be capable of circumventing detection. With advances in gene transfer vector design and therapeutic gene transfer, and demonstrations of safety and therapeutic benefit in humans, there is an increased probability of the pursuit of gene doping by athletes. In anticipation of the potential for gene doping, assays have been established to directly detect complementary DNA of genes that are top candidates for use in doping, as well as vector control elements. The development of molecular assays that are capable of exposing gene doping in sports can serve as a deterrent and may also identify athletes who have illicitly used gene transfer for performance enhancement. PCR-based methods to detect foreign DNA with high reliability, sensitivity, and specificity include TaqMan real-time PCR, nested PCR, and internal threshold control PCR.     

Hippocampal metabolomics using ultrahigh-resolution mass spectrometry reveals neuroinflammation from Alzheimer’s disease in CRND8 mice

In the wake of genomics, metabolomics characterizes the small molecular metabolites revealing the phenotypes induced by gene mutants. To address the metabolic signatures in the hippocampus of the amyloid-beta (Aβ) peptides produced in transgenic (Tg) CRND8 mice, high-field ion cyclotron resonance–Fourier transform mass spectrometry supported by LC-LTQ-Orbitrap was introduced to profile the extracted metabolites. More than 10,000 ions were detected in the mass profile for each sample. Subsequently, peak alignment and the 80 % rule followed by feature selection based on T score computation were performed. The putative identification was also conducted using the highly accurate masses with isotopic distribution by interfacing the MassTRIX database as well as MS/MS fragmentation generated in the LTQ-Orbitrap after chromatographic separation. Consequently, 58 differentiating masses were tentatively identified while up to 44 differentiating elemental compositions could not be biologically annotated in the databases. Nonetheless, of the putatively annotated masses, eicosanoids in arachidonic acid metabolism, fatty acid beta-oxidation disorders as well as disturbed glucose metabolism were highlighted as metabolic traits of Aβ toxicity in Tg CRND8 mice. Furthermore, a web-based bioinformatic tool was used for simulation of the metabolic pathways. As a result of the obtained metabolic signatures, the arachidonic acid metabolism dominates the metabolic perturbation in hippocampal tissues of Tg CRND8 mice compared to non-Tg littermates, indicating that Aβ toxicity functions neuroinflammation in hippocampal tissue and new theranostic opportunities might be offered by characterization of altered arachidonic acid metabolism for Alzheimer’s disease.      

Studies on Pyrrolidinones. An Improved One Pot Synthesis of 1,2,3,5,10,10a-Hexahydrobenz[f]indolizine-3,10-dione

Starting from the readily available N,O-bis trimethylsilyl pyroglutamate, an easy high yield one pot synthesis of ketone 1 was described.     

Synthesis of azafluorenones and related compounds using deprotocupration–aroylation followed by intramolecular direct arylation

The efficiency of the deprotocupration–aroylation of 2-chloropyridine using lithiocuprates prepared from CuX (X=Cl, Br) and LiTMP (TMP=2,2,6,6-tetramethylpiperidido, 2 equiv) was investigated. CuCl was identified as a more suitable copper source than CuBr for this purpose. Different diaryl ketones bearing a halogen at the 2 position of one of the aryl groups were synthesized in this way from azines and thiophenes. These were then involved in palladium-catalyzed ring closure: substrates underwent expected CH-activation-type arylation to afford fluorenone-type compounds, and were also subjected to cyclization reactions leading to xanthones, notably in the presence of oxygen-containing substituents or reagents.     

Lipophilic phthalocyanines for their potential interest in photodynamic therapy: synthesis and photo-physical properties

Several phthalocyanines with different peripheral substituents were prepared and characterized by MALDI-TOF, ¹H NMR, UV–vis, fluorescence, and singlet oxygen quantum yields and retention time in HPLC normal phase. Zinc was used as a central metal ion to increase the photodynamic therapy efficiency. Phthalonitrile or 4-nitro phthalonitriles were used as starting materials. The influence of lipophilicity on the photophysical and photochemical properties was evaluated.     

RGD-conjugated triarylmethyl radical as probe for electron paramagnetic imaging

A stable RGD-conjugated triarylmethyl (TAM) radical 3 has been synthesized in a straightforward three-step sequence. CT-03 ‘the Finland trityl’ which is a very popular contrast agent for electron paramagnetic resonance imaging has been covalently bound to the NH₂-ending of a pentapeptide commonly used to target integrins and confers tissue selectivity. Moreover, this new TAM radical is stable and sensitive to molecular oxygen.     

Raman spectroscopy and thermal analysis of gum and silica-filled NR/SBR blends prepared from latex system

Natural rubber/styrene-butadiene rubber (NR/SBR) blends, with and without silica, were prepared by co-coagulating the mixture of rubber latices and various amounts of well-dispersed silica suspension. An attempt to predict blend compositions was made using Raman spectroscopy in association with differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). It was found that the intensity of each Raman characteristic peak was strongly dependent on the blend composition, but there was no significant evolution with the presence of silica. Also, TGA results revealed an improvement in thermal stability of NR/SBR blends with increasing both SBR and silica contents due to the dilution effect. Two distinct glass transition temperatures (T_g) were observed in DSC thermograms of all blends, and their T_g values were independent on both blend composition and silica content. This indicated a physical blend formation, which agreed well with no shifts in Raman peaks of the blends in comparison with those of the individual rubbers. Linear regression with R² quality factor close to 0.99 was achieved when plotting intensity ratio at 1371/1302 cm^?1 versus blend ratios. On the other hand, the peak height ratio and heat capacity ratio from TGA and DSC analysis, respectively, yielded quadratic equations as a function of blend ratios.     

QMX: A versatile environment for hybrid calculations applied to the grafting of Al2Cl3Me3 on a silica surface

We present a new software to easily perform QM:MM and QM:QM' calculations called QMX. It follows the subtraction scheme and it is implemented in the Atomic Simulation Environment (ASE). Special attention is paid to couple molecular calculations with periodic boundaries approaches. QMX inherits the flexibility and versatility of the ASE package: any combination of methods namely force field, semiempirical, first principle, and ab initio, can be used as hybrid potential energy surface (PES). Its ease of use is demonstrated by considering the adsorption of Al₂Cl₃Me₃ on silica surface and by combining different levels of theory (from standard DFT to MP2 calculations) for the so‐called High Level cluster with standard PW91 density functional theory calculations for the Low Level environment. It is shown that the High Level cluster must contain the silanol group close to the aluminum atoms. The bridging adsorption is favored by 58 kJ mol^?1 at the MP2:PW91 level with respect to the terminal position. Using large clusters at the MP2:PW91 level, it is shown that PW91 calculations are sufficient for structure optimization but that embedded methods are required for accurate energy profiles. © 2013 Wiley Periodicals, Inc.     

Laterally dibenzyloxy-branched nematogens with large nematic range and rich solid polymorphism

Mesogenic compounds containing four rings in the core usually have very high melting points. However, when two identical lateral benzyloxy groups are introduced on the same side of one of the central rings, the melting point is lowered dramatically and a large nematic range is retained. This range is affected by the bulkiness of the para-substituents in the lateral rings. Methyl groups can be introduced in the ortho- or meta-positions with a consequent decrease in the melting temperature without much affecting the nematic range. These compounds exhibit a rich solid polymorphism which is certainly related to the effect of the conformations of the lateral substituent on the molecular arrangment in the solid phase. Some preliminary NMR experiments on the nematic phase indicate that the molecular long axis coincides with the core axis, whereas the para-axis of the lateral fragment makes an angle close to the magic angle with respect to the molecular long axis.