AlphaII-spectrin participates in the surface expression of cell adhesion molecule L1 and neurite outgrowth

AlphaII-spectrin, a basic component of the spectrin-based scaffold which organizes and stabilizes membrane microdomains in most animal cells, has been recently implicated in cell adherence and actin dynamics. Here we investigated the contribution of αΙΙ-spectrin to neuritogenesis, a highly complex cellular process which requires continuous actin cytoskeleton remodeling and cross-talk between extracellular cues and their cell surface receptors, including cell adhesion molecules. Using RNA interference-mediated gene silencing to down-regulate αΙΙ-spectrin expression in human neuroblastoma SH-SY5Y cells, we observed major changes in neurite morphology and cell shape: (1) reduced mean length and a higher number of neurites per cell; occasional long neurites were thinner and displayed abnormal adhesiveness during cell migration resulting in frequent breaks; similar persisting adhesiveness and breaks were also observed in trailing edges of cell bodies; (2) irregular polygonal cell shape in parallel with loss of cortical F-actin from neuronal cell bodies; (3) reduction in protein levels of αΙ- and βΙ-spectrins, but not βΙΙ-spectrin (4) decreased global expression of adhesion molecule L1 and spectrin-binding adapter ankyrin-B, which links L1 to the plasma membrane. Remarkably, αΙΙ-spectrin depletion affected L1 – but not NCAM – cell surface expression, and L1 clustering at growth cones. This study demonstrates that αΙΙ-spectrin is implicated in normal morphology and adhesive properties of neuron cell bodies and neurites, and in cell surface expression and organization of adhesion molecule L1.     

Enteral glutamine infusion modulates ubiquitination of heat shock proteins,Grp-75 and Apg-2, in the human duodenal mucosa

Glutamine, the most abundant amino acid in the human body, plays several important roles in the intestine. Previous studies showed that glutamine may affect protein expression by regulating ubiquitin–proteasome system. We thus aimed to evaluate the effects of gl utamine on ubiquitinated proteins in human duodenal mucosa. Five healthy male volunteers were included and received during 5 h, on two occasions and in a random order, either an enteral infusion of maltodextrins alone (0.25 g kg^?1 h^?1, control), mimicking carbohydrate-fed state, or maltodextrins with glutamine (0.117 g kg^?1 h^?1, glutamine). Endoscopic duodenal biopsies were then taken. Total cellular protein extracts were separated by 2D gel electrophoresis and analyzed by an immunodetection using anti-ubiquitin antibody. Differentially ubiquitinated proteins were then identified by liquid chromatography–electrospray ionization MS/MS. Five proteins were differentially ubiquitinated between control and glutamine conditions. Among these proteins, we identified two chaperone proteins, Grp75 and hsp74. Grp75 was less ubiquitinated after glutamine infusion compared with control. In contrast, hsp74, also called Apg-2, was more ubiquitinated after glutamine. In conclusion, we provide evidence that glutamine may regulate ubiquitination processes of specific proteins, i.e., Grp75 and Apg-2. Grp75 has protective and anti-inflammatory properties, while Apg-2 indirectly regulates stress-induced cell survival and proliferation through interaction with ZO-1. Further studies should confirm these results in stress conditions.     

Quantum State Engineering of Light with Continuous-wave Optical Parametric Oscillators

Engineering non-classical states of the electromagnetic field is a central quest for quantum optics^1,2. Beyond their fundamental significance, such states are indeed the resources for implementing various protocols, ranging from enhanced metrology to quantum communication and computing. A variety of devices can be used to generate non-classical states, such as single emitters, light-matter interfaces or non-linear systems³. We focus here on the use of a continuous-wave optical parametric oscillator^3,4. This system is based on a non-linear χ² crystal inserted inside an optical cavity and it is now well-known as a very efficient source of non-classical light, such as single-mode or two-mode squeezed vacuum depending on the crystal phase matching. Squeezed vacuum is a Gaussian state as its quadrature distributions follow a Gaussian statistics. However, it has been shown that number of protocols require non-Gaussian states⁵. Generating directly such states is a difficult task and would require strong χ³ non-linearities. Another procedure, probabilistic but heralded, consists in using a measurement-induced non-linearity via a conditional preparation technique operated on Gaussian states. Here, we detail this generation protocol for two non-Gaussian states, the single-photon state and a superposition of coherent states, using two differently phase-matched parametric oscillators as primary resources. This technique enables achievement of a high fidelity with the targeted state and generation of the state in a well-controlled spatiotemporal mode.     

Drosophila growth and development: Keeping things in proportion

Comment on: Colombani J, et al. Science 2012; 336:582-5.     

A Peptidoglycan Fragment Triggers β-lactam Resistance in Bacillus licheniformis

To resist to β-lactam antibiotics Eubacteria either constitutively synthesize a β-lactamase or a low affinity penicillin-binding protein target, or induce its synthesis in response to the presence of antibiotic outside the cell. In Bacillus licheniformis and Staphylococcus aureus, a membrane-bound penicillin receptor (BlaR/MecR) detects the presence of β-lactam and launches a cytoplasmic signal leading to the inactivation of BlaI/MecI repressor, and the synthesis of a β-lactamase or a low affinity target. We identified a dipeptide, resulting from the peptidoglycan turnover and present in bacterial cytoplasm, which is able to directly bind to the BlaI/MecI repressor and to destabilize the BlaI/MecI-DNA complex. We propose a general model, in which the acylation of BlaR/MecR receptor and the cellular stress induced by the antibiotic, are both necessary to generate a cell wall-derived coactivator responsible for the expression of an inducible β-lactam-resistance factor. The new model proposed confirms and emphasizes the role of peptidoglycan degradation fragments in bacterial cell regulation.     

Structural and Enzymatic characterization of the lactonase SisLac from Sulfolobus islandicus

Background

A new member of the Phosphotriesterase-Like Lactonases (PLL) family from the hyperthermophilic archeon Sulfolobus islandicus (SisLac) has been characterized. SisLac is a native lactonase that exhibits a high promiscuous phosphotriesterase activity. SisLac thus represents a promising target for engineering studies, exhibiting both detoxification and bacterial quorum quenching abilities, including human pathogens such as Pseudomonas aeruginosa.

Methodology/Principal Findings

Here, we describe the substrate specificity of SisLac, providing extensive kinetic studies performed with various phosphotriesters, esters, N-acyl-homoserine lactones (AHLs) and other lactones as substrates. Moreover, we solved the X-ray structure of SisLac and structural comparisons with the closely related SsoPox structure highlighted differences in the surface salt bridge network and the dimerization interface. SisLac and SsoPox being close homologues (91% sequence identity), we undertook a mutational study to decipher these structural differences and their putative consequences on the stability and the catalytic properties of these proteins.

Conclusions/Significance

We show that SisLac is a very proficient lactonase against aroma lactones and AHLs as substrates. Hence, data herein emphasize the potential role of SisLac as quorum quenching agent in Sulfolobus. Moreover, despite the very high sequence homology with SsoPox, we highlight key epistatic substitutions that influence the enzyme stability and activity.     

Normalisation to blood activity is required for the accurate quantification of Na/I symporter ectopic expression by SPECT/CT in individual subjects

The utilisation of the Na/I symporter (NIS) and associated radiotracers as a reporter system for imaging gene expression is now reaching the clinical setting in cancer gene therapy applications. However, a formal assessment of the methodology in terms of normalisation of the data still remains to be performed, particularly in the context of the assessment of activities in individual subjects in longitudinal studies. In this context, we administered to mice a recombinant, replication-incompetent adenovirus encoding rat NIS, or a human colorectal carcinoma cell line (HT29) encoding mouse NIS. We used (99m)Tc pertechnetate as a radiotracer for SPECT/CT imaging to determine the pattern of ectopic NIS expression in longitudinal kinetic studies. Some animals of the cohort were culled and NIS expression was measured by quantitative RT-PCR and immunohistochemistry. The radioactive content of some liver biopsies was also measured ex vivo. Our results show that in longitudinal studies involving datasets taken from individual mice, the presentation of non-normalised data (activity expressed as %ID/g or %ID/cc) leads to 'noisy', and sometimes incoherent, results. This variability is due to the fact that the blood pertechnetate concentration can vary up to three-fold from day to day. Normalisation of these data with blood activities corrects for these inconsistencies. We advocate that, blood pertechnetate activity should be determined and used to normalise the activity measured in the organ/region of interest that expresses NIS ectopically. Considering that NIS imaging has already reached the clinical setting in the context of cancer gene therapy, this normalisation may be essential in order to obtain accurate and predictive information in future longitudinal clinical studies in biotherapy.     

Open Source Tracking and Analysis of Adult Drosophila Locomotion in Buridan's Paradigm with and without Visual Targets

Background

Insects have been among the most widely used model systems for studying the control of locomotion by nervous systems. In Drosophila, we implemented a simple test for locomotion: in Buridan''s paradigm, flies walk back and forth between two inaccessible visual targets [1]. Until today, the lack of easily accessible tools for tracking the fly position and analyzing its trajectory has probably contributed to the slow acceptance of Buridan''s paradigm.

Methodology/Principal Findings

We present here a package of open source software designed to track a single animal walking in a homogenous environment (Buritrack) and to analyze its trajectory. The Centroid Trajectory Analysis (CeTrAn) software is coded in the open source statistics project R. It extracts eleven metrics and includes correlation analyses and a Principal Components Analysis (PCA). It was designed to be easily customized to personal requirements. In combination with inexpensive hardware, these tools can readily be used for teaching and research purposes. We demonstrate the capabilities of our package by measuring the locomotor behavior of adult Drosophila melanogaster (whose wings were clipped), either in the presence or in the absence of visual targets, and comparing the latter to different computer-generated data. The analysis of the trajectories confirms that flies are centrophobic and shows that inaccessible visual targets can alter the orientation of the flies without changing their overall patterns of activity.

Conclusions/Significance

Using computer generated data, the analysis software was tested, and chance values for some metrics (as well as chance value for their correlation) were set. Our results prompt the hypothesis that fixation behavior is observed only if negative phototaxis can overcome the propensity of the flies to avoid the center of the platform. Together with our companion paper, we provide new tools to promote Open Science as well as the collection and analysis of digital behavioral data.