Interfacial kinetic and binding properties of the complete set of human and mouse groups I,II, V,X, and XII secreted phospholipases A2

Expression of the full set of human and mouse groups I, II, V, X, and XII secreted phospholipases A(2) (sPLA(2)s) in Escherichia coli and insect cells has provided pure recombinant enzymes for detailed comparative interfacial kinetic and binding studies. The set of mammalian sPLA(2)s display dramatically different sensitivity to dithiothreitol. The specific activity for the hydrolysis of vesicles of differing phospholipid composition by these enzymes varies by up to 4 orders of magnitude, and yet all enzymes display similar catalytic site specificity toward phospholipids with different polar head groups. Discrimination between sn-2 polyunsaturated versus saturated fatty acyl chains is <6-fold. These enzymes display apparent dissociation constants for activation by calcium in the 1-225 microm range, depending on the phospholipid substrate. Analysis of the inhibition by a set of 12 active site-directed, competitive inhibitors reveals a large variation in the potency among the mammalian sPLA(2)s, with Me-Indoxam being the most generally potent sPLA(2) inhibitor. A dramatic correlation exists between the ability of the sPLA(2)s to hydrolyze phosphatidylcholine-rich vesicles efficiently in vitro and the ability to release arachidonic acid when added exogenously to mammalian cells; the group V and X sPLA(2)s are uniquely efficient in this regard.     

Brain-inspired model for early vocal learning and correspondence matching using free-energy optimization

We propose a developmental model inspired by the cortico-basal system (CX-BG) for vocal learning in babies and for solving the correspondence mismatch problem they face when they hear unfamiliar voices, with different tones and pitches. This model is based on the neural architecture INFERNO standing for Iterative Free-Energy Optimization of Recurrent Neural Networks. Free-energy minimization is used for rapidly exploring, selecting and learning the optimal choices of actions to perform (eg sound production) in order to reproduce and control as accurately as possible the spike trains representing desired perceptions (eg sound categories). We detail in this paper the CX-BG system responsible for linking causally the sound and motor primitives at the order of a few milliseconds. Two experiments performed with a small and a large audio database show the capabilities of exploration, generalization and robustness to noise of our neural architecture in retrieving audio primitives during vocal learning and during acoustic matching with unheared voices (different genders and tones).     

Binding Mechanism of the Peptidoglycan Hydrolase Acm2: Low Affinity,Broad Specificity

Peptidoglycan hydrolases are bacterial secreted enzymes that cleave covalent bonds in the cell-wall peptidoglycan, thereby fulfilling major physiological functions during cell growth and division. Although the molecular structure and functional roles of these enzymes have been widely studied, the molecular details underlying their interaction with peptidoglycans remain largely unknown, mainly owing to the paucity of appropriate probing techniques. Here, we use atomic force microscopy to explore the binding mechanism of the major autolysin Acm2 from the probiotic bacterium Lactobacillus plantarum. Atomic force microscopy imaging shows that incubation of bacterial cells with Acm2 leads to major alterations of the cell-surface nanostructure, leading eventually to cell lysis. Single-molecule force spectroscopy demonstrates that the enzyme binds with low affinity to structurally different peptidoglycans and to chitin, and that glucosamine in the glycan chains is the minimal binding motif. We also find that Acm2 recognizes mucin, the main extracellular component of the intestinal mucosal layer, thereby suggesting that this enzyme may also function as a cell adhesion molecule. The binding mechanism (low affinity and broad specificity) of Acm2 may represent a generic mechanism among cell-wall hydrolases for guiding cell division and cell adhesion.     

Rapid detection of carbapenem resistance in Acinetobacter baumannii using matrix-assisted laser desorption ionization-time of flight mass spectrometry

Rapid detection of carbapenem-resistant Acinetobacter baumannii strains is critical and will benefit patient care by optimizing antibiotic therapies and preventing outbreaks. Herein we describe the development and successful application of a mass spectrometry profile generated by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) that utilized the imipenem antibiotic for the detection of carbapenem resistance in a large series of A. baumannii clinical isolates from France and Algeria. A total of 106 A. baumannii strains including 63 well-characterized carbapenemase-producing and 43 non-carbapenemase-producing strains, as well as 43 control strains (7 carbapenem-resistant and 36 carbapenem-sensitive strains) were studied. After an incubation of bacteria with imipenem for up to 4 h, the mixture was centrifuged and the supernatant analyzed by MALDI-TOF MS. The presence and absence of peaks representing imipenem and its natural metabolite was analyzed. The result was interpreted as positive for carbapenemase production if the specific peak for imipenem at 300.0 m/z disappeared during the incubation time and if the peak of the natural metabolite at 254.0 m/z increased as measured by the area under the curves leading to a ratio between the peak for imipenem and its metabolite being <0.5. This assay, which was applied to the large series of A. baumannii clinical isolates, showed a sensitivity of 100.0% and a specificity of 100.0%. Our study is the first to demonstrate that this quick and simple assay can be used as a routine tool as a point-of-care method for the identification of A. baumannii carbapenemase-producers in an effort to prevent outbreaks and the spread of uncontrollable superbugs.     

Design and synthesis of bridged piperidine and piperazine isosteres

We have developed versatile methods toward the synthesis of a variety of piperidine/piperazine bridged isosteres of pridopidine. The compounds were assessed against the D2 receptor in agonist and antagonist modes and against the D4 receptor in agonist mode. hERG Binding and the ADME profiles were studied.     

Cockayne syndrome group B protein regulates fork restart,fork progression and MRE11-dependent fork degradation in BRCA1/2-deficient cells

Cockayne syndrome group B (CSB) protein has been implicated in the repair of a variety of DNA lesions that induce replication stress. However, little is known about its role at stalled replication forks. Here, we report that CSB is recruited to stalled forks in a manner dependent upon its T1031 phosphorylation by CDK. While dispensable for MRE11 association with stalled forks in wild-type cells, CSB is required for further accumulation of MRE11 at stalled forks in BRCA1/2-deficient cells. CSB promotes MRE11-mediated fork degradation in BRCA1/2-deficient cells. CSB possesses an intrinsic ATP-dependent fork reversal activity in vitro, which is activated upon removal of its N-terminal region that is known to autoinhibit CSB’s ATPase domain. CSB functions similarly to fork reversal factors SMARCAL1, ZRANB3 and HLTF to regulate slowdown in fork progression upon exposure to replication stress, indicative of a role of CSB in fork reversal in vivo. Furthermore, CSB not only acts epistatically with MRE11 to facilitate fork restart but also promotes RAD52-mediated break-induced replication repair of double-strand breaks arising from cleavage of stalled forks by MUS81 in BRCA1/2-deficient cells. Loss of CSB exacerbates chemosensitivity in BRCA1/2-deficient cells, underscoring an important role of CSB in the treatment of cancer lacking functional BRCA1/2.     

Regulation of ppk expression and in vivo function of Ppk in Streptomyces lividans TK24

The ppk gene of Streptomyces lividans encodes an enzyme catalyzing, in vitro, the reversible polymerization of the gamma phosphate of ATP into polyphosphate and was previously shown to play a negative role in the control of antibiotic biosynthesis (H. Chouayekh and M. J. Virolle, Mol. Microbiol. 43:919-930, 2002). In the present work, some regulatory features of the expression of ppk were established and the polyphosphate content of S. lividans TK24 and the ppk mutant was determined. In Pi sufficiency, the expression of ppk was shown to be low but detectable. DNA gel shift experiments suggested that ppk expression might be controlled by a repressor using ATP as a corepressor. Under these conditions, short acid-soluble polyphosphates accumulated upon entry into the stationary phase in the wild-type strain but not in the ppk mutant strain. The expression of ppk under Pi-limiting conditions was shown to be much higher than that under Pi-sufficient conditions and was under positive control of the two-component system PhoR/PhoP. Under these conditions, the polyphosphate content of the cell was low and polyphosphates were reproducibly found to be longer and more abundant in the ppk mutant strain than in the wild-type strain, suggesting that Ppk might act as a nucleoside diphosphate kinase. In light of our results, a novel view of the role of this enzyme in the regulation of antibiotic biosynthesis in S. lividans TK24 is proposed.     

Synthesis,crystal structure and antibacterial activity of new highly functionalized ionic compounds based on the imidazole nucleus

Several new highly functionalized imidazolium derivatives were synthesized, via appropriate synthetic routes, using imidazole, 1-methylimidazole and 2-phenyl-1-methylimidazole as key intermediates. The antibacterial activity of the prepared compounds was evaluated against: Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella thipymurium using disk-diffusion and MIC methods. Crystal X-ray structures are reported for six compounds.     

Should Metabolic Diseases Be Systematically Screened in Nonsyndromic Autism Spectrum Disorders?

Background

In the investigation of autism spectrum disorders (ASD), a genetic cause is found in approximately 10–20%. Among these cases, the prevalence of the rare inherited metabolic disorders (IMD) is unknown and poorly evaluated. An IMD responsible for ASD is usually identified by the associated clinical phenotype such as dysmorphic features, ataxia, microcephaly, epilepsy, and severe intellectual disability (ID). In rare cases, however, ASD may be considered as nonsyndromic at the onset of a related IMD.

Objectives

To evaluate the utility of routine metabolic investigations in nonsyndromic ASD.

Patients and Methods

We retrospectively analyzed the results of a metabolic workup (urinary mucopolysaccharides, urinary purines and pyrimidines, urinary creatine and guanidinoacetate, urinary organic acids, plasma and urinary amino acids) routinely performed in 274 nonsyndromic ASD children.

Results

The metabolic parameters were in the normal range for all but 2 patients: one with unspecific creatine urinary excretion and the other with persistent 3-methylglutaconic aciduria.

Conclusions

These data provide the largest ever reported cohort of ASD patients for whom a systematic metabolic workup has been performed; they suggest that such a routine metabolic screening does not contribute to the causative diagnosis of nonsyndromic ASD. They also emphasize that the prevalence of screened IMD in nonsyndromic ASD is probably not higher than in the general population (<0.5%). A careful clinical evaluation is probably more reasonable and of better medical practice than a costly systematic workup.