827Spatio-Temporal Quantification of FRET in Living Cells by Fast Time-Domain FLIM: A Comparative Study of Non-Fitting Methods

Förster Resonance Energy Transfer (FRET) measured with Fluorescence Lifetime Imaging Microscopy (FLIM) is a powerful technique to investigate spatio-temporal regulation of protein-protein interactions in living cells. When using standard fitting methods to analyze time domain FLIM, the correct estimation of the FRET parameters requires a high number of photons and therefore long acquisition times which are incompatible with the observation of dynamic protein-protein interactions. Recently, non-fitting strategies have been developed for the analysis of FLIM images: the polar plot or “phasor” and the minimal fraction of interacting donor mf_D. We propose here a novel non-fitting strategy based on the calculation of moments. We then compare the performance of these three methods when shortening the acquisition time: either by reducing the number of counted photons N or the number of temporal channels N_ch, which is particularly adapted for the original fast-FLIM prototype presented in this work that employs the time gated approach. Based on theoretical calculations, Monte Carlo simulations and experimental data, we determine the domain of validity of each method. We thus demonstrate that the polar approach remains accurate for a large range of conditions (low N, N_ch or small fractions of interacting donor f_D). The validity domain of the moments method is more restricted (not applicable when f_D<0.25 or when N_ch = 4) but it is more precise than the polar approach. We also demonstrate that the mf_D is robust in all conditions and it is the most precise strategy; although it does not strictly provide the fraction of interacting donor. We show using the fast-FLIM prototype (with an acquisition rate up to 1 Hz) that these non-fitting strategies are very powerful for on-line analysis on a standard computer and thus for quantifying automatically the spatio-temporal activation of Rac-GTPase in living cells by FRET.     

A readily usable two‐photon fluorescence lifetime microendoscope

A two‐photon fluorescence lifetime (2P‐FLIM) microendoscope, capable of energetic metabolism imaging through the intracellular nicotinamide adenine dinucleotide (NADH) autofluorescence, at sub‐cellular resolution, is demonstrated. It exhibits readily usable characteristics such as convenient endoscope probe diameter (≈2 mm), fiber length (>5 m) and data accumulation rate (16 frames per second (fps)), leading to a FLIM refreshing rate of ≈0.1 to 1 fps depending on the sample. The spiral scanning image formation does not influence the instrument response function (IRF) characteristics of the system. Near table‐top microscope performances are achieved through a comprehensive system including a home‐designed spectro‐temporal pulse shaper and a custom air‐silica double‐clad photonic crystal fiber, which enables to reach up to 40 mW of ≈100 fs pulses @ 760 nm with a 80 MHz repetition rate. A GRadient INdex (GRIN) lens provides a lateral resolution of 0.67 μm at the focus of the fiber probe. Intracellular NADH fluorescence lifetime data are finally acquired on cultured cells at 16 fps.      

CFTR Protects against Mycobacterium abscessus Infection by Fine-Tuning Host Oxidative Defenses

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Evaluation of the lambda model for human postural control during ankle strategy

An accurate modeling of human stance might be helpful in assessing postural deficit. The objective of this article is to validate a mathematical postural control model for quiet standing posture. The postural dynamics is modeled in the sagittal plane as an inverted pendulum with torque applied at the ankle joint. The torque control system is represented by the physiological lambda model. Two neurophysiological command variables of the central nervous system, designated and , establish the dynamic threshold muscle at which motoneuron recruitment begins. Kinematic data and electromyographic signals were collected on four young males in order to measure small voluntary sway and quiet standing posture. Validation of the mathematical model was achieved through comparison of the experimental and simulated results. The mathematical model allows computation of the unmeasurable neurophysiological commands and that control the equilibrium position and stability. Furthermore, with the model it is possible to conclude that low-amplitude body sway during quiet stance is commanded by the central nervous system.     

Treatment of complex interphalangeal joint fractures with dynamic external traction: a series of 20 cases

Data are reported for a series of 20 patients who were treated with the pins and rubbers traction system for fractures of the proximal interphalangeal joints of the long fingers. This technique allows fracture reduction with external dynamic traction and immediate active mobilization. Two patients in the series were lost to follow-up monitoring. For two others, the pins and rubbers traction system needed to be removed early (during the first week) because of intolerance or infection. Sixteen patients who were reexamined after minimal follow-up periods of 1 year demonstrated a mean active range of motion of 85.9 degrees for the injured joint; only one patient experienced intermittent pain.     

Adjacent and spontaneous neurotization after distal digital replantation in children

In an exclusively pediatric population, this retrospective study examined the functional and aesthetic results after distal replantation without nerve suture. The aim was to demonstrate, in the child, the presence of spontaneous nervous regeneration resulting in a fingertip pulp with discriminatory sensation. Eight amputations in eight children with a mean age of 9 years and 2 months on the day of the accident were reviewed. The cases were managed by a single surgeon over a period of 8 years and were collected from two different hand centers. The patients were then examined by a different surgeon, and the data were collected. Sensibility was evaluated using the Weber, Semmes-Weinstein, and wrinkle tests. The results were excellent, with mean values of 4.6 mm for the Weber test, 3.3 for the Semmes-Weinstein test, and a positive wrinkle test in all subjects. All patients thus recovered discriminatory sensation with minimal aesthetic sequelae. The usual factors adversely affecting the results of the replantation (ischemic time, level and mechanism of the amputation, and quality of the venous return) were examined, but no statistical analysis was performed because of the small sample size. This study demonstrates the presence of the clinical phenomenon of adjacent neurotization in the absence of nerve repair. It thus confirms that children are excellent candidates for replantation of the distal extremities, even when nerve suture is not performed.     

ARIA2: automated NOE assignment and data integration in NMR structure calculation

Modern structural genomics projects demand for integrated methods for the interpretation and storage of nuclear magnetic resonance (NMR) data. Here we present version 2.1 of our program ARIA (Ambiguous Restraints for Iterative Assignment) for automated assignment of nuclear Overhauser enhancement (NOE) data and NMR structure calculation. We report on recent developments, most notably a graphical user interface, and the incorporation of the object-oriented data model of the Collaborative Computing Project for NMR (CCPN). The CCPN data model defines a storage model for NMR data, which greatly facilitates the transfer of data between different NMR software packages. Availability: A distribution with the source code of ARIA 2.1 is freely available at http://www.pasteur.fr/recherche/unites/Binfs/aria2.