InferenceMAP: Mapping of Single-Molecule Dynamics with Bayesian Inference

Single-particle tracking (SPT) grants unprecedented insight into cellular function at the molecular scale [1]. Throughout the cell, the movement of single-molecules is generally heterogeneous and complex. Hence, there is an imperative to understand the multi-scale nature of single-molecule dynamics in biological systems. We have previously shown that with high-density SPT, spatial maps of the parameters that dictate molecule motion can be generated to intricately describe cellular environments [2,3,4]. To date, however, there exist no publically available tools that reconcile trajectory data to generate the aforementioned maps. We address this void in the SPT community with InferenceMAP: an interactive software package that uses a powerful Bayesian method to map the dynamic cellular space experienced by individual biomolecules.Comment: 56 page

From Single-Molecule Interactions to Population-Level Dynamics: Understanding the Complex Organization of RNA Pol II in the Nucleus of Living Cells

Transcription involves a complex exchange within a reservoir of proteins in the nucleoplasm, and the specific recruitment of individual proteins at specific gene loci. However, understanding the spatial distribution of individual proteins and the temporal behavior in the nucleus of living cells remains challenging. Using 3D super-resolution fluorescence microscopy and cluster analysis, we observe that the distribution of RNA Polymerase II (Pol II) cluster sizes, measured as the number of polymerases per cluster, follows a −3/2 power law. Radial dependent analysis of the spatial distribution of Pol II also shows scale-invariance, consistent with a so-called self-organized criticality in a fractal geometry of dimension ∼2.7. These results suggest a diffusion-based mechanism whereby, via transient interactions, massive recruitment and dismissal of pol II molecules can occur at specific loci in the nucleoplasm. Kinetic measurements using single-molecule detection in live cells reveal Pol II binding dynamics within minutes. Serum-induced transcription increased Pol II binding kinetics in live cells by an order of magnitude. Together, these results provide a comprehensive view of the spatio-temporal organization of Pol II in the nucleus: from the global population distribution, to single molecule recruitment at specific loci in live cells. This comprehensive single-cell approach can be adopted for other proteins beside RNA Pol II, for real-time quantification of protein organization in vivo, with single-molecule sensitivity

Global overview of the management of acute cholecystitis during the COVID-19 pandemic (CHOLECOVID study)

Background: This study provides a global overview of the management of patients with acute cholecystitis during the initial phase of the COVID-19 pandemic. Methods: CHOLECOVID is an international, multicentre, observational comparative study of patients admitted to hospital with acute cholecystitis during the COVID-19 pandemic. Data on management were collected for a 2-month study interval coincident with the WHO declaration of the SARS-CoV-2 pandemic and compared with an equivalent pre-pandemic time interval. Mediation analysis examined the influence of SARS-COV-2 infection on 30-day mortality. Results: This study collected data on 9783 patients with acute cholecystitis admitted to 247 hospitals across the world. The pandemic was associated with reduced availability of surgical workforce and operating facilities globally, a significant shift to worse severity of disease, and increased use of conservative management. There was a reduction (both absolute and proportionate) in the number of patients undergoing cholecystectomy from 3095 patients (56.2 per cent) pre-pandemic to 1998 patients (46.2 per cent) during the pandemic but there was no difference in 30-day all-cause mortality after cholecystectomy comparing the pre-pandemic interval with the pandemic (13 patients (0.4 per cent) pre-pandemic to 13 patients (0.6 per cent) pandemic; P = 0.355). In mediation analysis, an admission with acute cholecystitis during the pandemic was associated with a non-significant increased risk of death (OR 1.29, 95 per cent c.i. 0.93 to 1.79, P = 0.121). Conclusion: CHOLECOVID provides a unique overview of the treatment of patients with cholecystitis across the globe during the first months of the SARS-CoV-2 pandemic. The study highlights the need for system resilience in retention of elective surgical activity. Cholecystectomy was associated with a low risk of mortality and deferral of treatment results in an increase in avoidable morbidity that represents the non-COVID cost of this pandemic

Planar Photonic Crystals for Biosensing

In this thesis, planar photonic crystals for optofluidic biosensing applications are analyzed. Planar photonic crystals are optically resonant structures which possess modal characteristics which can be exploited for biosensing applications. Sensing is achieved by detecting changes in refractive index due to analyte interactions in a sampled fluid. This work describes a broad study of photonic crystal slab sensors, with special consideration to biosensing. Outlined are considerations pertaining to sensing figures of merit, device fabrication, and performance. Results of simulations and device characterization indicate that planar photonic crystals possess sensing attributes similar or better than existing optically resonant refractive index sensors, such as surface plasmon resonance, grating, and interferometric waveguide sensors. Additionally, these photonic crystals can be patterned in large-areas which enable a simple light coupling scheme. All considered, their appeal as a biosensing solution is justified in the area of in vitro diagnostics.MAS

Vers une cartographie cellule entière de molécule unique dynamique

Imaging of single molecules inside living cells confers insight to biological function at its most granular level. Single molecules experience a nanoscopic environment that is complicated, and in general, poorly understood. The modality of choice for probing this environment is live-cell localisation microscopy, where trajectories of single molecules can be captured. For many years, the great stumbling block in comprehension of physical processes at this scale was the lack of information accessible; statistical significance and robust assertions are hardly possible from a few dozen trajectories. It is the onset of high-density single-particle tracking that has dramatically reframed the possibilities of such studies. Importantly, the consequential amounts of data it provides invites the use of powerful statistical tools that assign probabilistic descriptions to experimental observations. In this thesis, Bayesian inference tools have been developed to elucidate the behaviour of single molecules via the mapping of motion parameters. As a readout, maps describe heterogeneities at local and whole-cell scales. Importantly, they grant quantitative details into basic cellular processes. This thesis uses the mapping approach to study receptor-scaffold interactions inside neurons and non-neuronal cells. A promising system in which interactions are patterned is also examined. It is shown that interactions of different types of chimeric glycine receptors to the gephyrin scaffold protein may be described and distinguished in situ. Finally, the prospects of whole-cell mapping in three-dimensions are evaluated based on a discussion of state-of-the-art volumetric microscopy techniques.La compréhension fondamentale de fonctions biologiques est accordée par l’imagerie de molécules uniques dans les cellules vivantes. Cet environnement nanoscopique est compliqué et largement mal compris. La microscopie de localisation permet cet environnement d’être sondé avec le suivi de molécules uniques. Depuis longtemps, l’obstacle principal qui empêcher la compréhension de processus physiques à cette échelle était la pénurie d’information accessible; de fortes hypothèses ne peuvent pas être établies à partir de quelques dizaines de trajectoires de molécules uniques. L’introduction des techniques de suivi de molécules à haute densité a recadré les possibilités. Dans cette thèse, les outils d’inférence Bayesienne ont été développé pour élucider le comportement de molécules uniques à partir la cartographie de leurs paramètres physiques de mouvement. Ces cartes décrivent, entre autre, les hétérogénéités aux échelles locales mais aussi à l’échelle de la cellule entière. Notamment elles dévoilent les détails quantitatifs sur les processus cellulaires de base. En utilisant cette approche cartographique, les interactions entre récepteurs et protéines d’échafaudage chez les neurones et les cellules non-neuronales sont étudiés. Une étude sur les interactions imprimées dans la cellule est également effectuée grace à un système prometteur d’impression de protéine. Les différents types d’interactions entre constructions chimériques du récepteur de glycine et de protéines d’échafaudage de géphyrine sont décrits et distingué in situ. Enfin, les perspectives vis-à-vis l’obtention de cartes tridimensionnelles de dynamiques de molécules uniques sont également commentées

Vers une cartographie cellule entière de molécule unique dynamique

La compréhension fondamentale de fonctions biologiques est accordée par l’imagerie de molécules uniques dans les cellules vivantes. Cet environnement nanoscopique est compliqué et largement mal compris. La microscopie de localisation permet cet environnement d’être sondé avec le suivi de molécules uniques. Depuis longtemps, l’obstacle principal qui empêcher la compréhension de processus physiques à cette échelle était la pénurie d’information accessible; de fortes hypothèses ne peuvent pas être établies à partir de quelques dizaines de trajectoires de molécules uniques. L’introduction des techniques de suivi de molécules à haute densité a recadré les possibilités. Dans cette thèse, les outils d’inférence Bayesienne ont été développé pour élucider le comportement de molécules uniques à partir la cartographie de leurs paramètres physiques de mouvement. Ces cartes décrivent, entre autre, les hétérogénéités aux échelles locales mais aussi à l’échelle de la cellule entière. Notamment elles dévoilent les détails quantitatifs sur les processus cellulaires de base. En utilisant cette approche cartographique, les interactions entre récepteurs et protéines d’échafaudage chez les neurones et les cellules non-neuronales sont étudiés. Une étude sur les interactions imprimées dans la cellule est également effectuée grace à un système prometteur d’impression de protéine. Les différents types d’interactions entre constructions chimériques du récepteur de glycine et de protéines d’échafaudage de géphyrine sont décrits et distingué in situ. Enfin, les perspectives vis-à-vis l’obtention de cartes tridimensionnelles de dynamiques de molécules uniques sont également commentées.Imaging of single molecules inside living cells confers insight to biological function at its most granular level. Single molecules experience a nanoscopic environment that is complicated, and in general, poorly understood. The modality of choice for probing this environment is live-cell localisation microscopy, where trajectories of single molecules can be captured. For many years, the great stumbling block in comprehension of physical processes at this scale was the lack of information accessible; statistical significance and robust assertions are hardly possible from a few dozen trajectories. It is the onset of high-density single-particle tracking that has dramatically reframed the possibilities of such studies. Importantly, the consequential amounts of data it provides invites the use of powerful statistical tools that assign probabilistic descriptions to experimental observations. In this thesis, Bayesian inference tools have been developed to elucidate the behaviour of single molecules via the mapping of motion parameters. As a readout, maps describe heterogeneities at local and whole-cell scales. Importantly, they grant quantitative details into basic cellular processes. This thesis uses the mapping approach to study receptor-scaffold interactions inside neurons and non-neuronal cells. A promising system in which interactions are patterned is also examined. It is shown that interactions of different types of chimeric glycine receptors to the gephyrin scaffold protein may be described and distinguished in situ. Finally, the prospects of whole-cell mapping in three-dimensions are evaluated based on a discussion of state-of-the-art volumetric microscopy techniques

Mechanisms of anaesthetic depression of neocortical arousal

The most widely accepted hypotheses suggest that general anaesthetics interrupt conscious processes in the brain by decreasing synaptic excitation or by potentiating synaptic inhibition, especially in the neocortex. The putative transmitters in the neurological systems that generate neocortical arousal include acetylcholine, glutamate and γ-aminobutyrate (GABA). The primary objective here was to determine the neuronal mechanisms by which anaesthetics may obtund this arousal. The majority of the investigations were carried out on pyramidal neurons in layers IV and V of guinea pig neocortex (in vitro slices), using intracellular recording and pharmacological, including microiontophoretic, techniques. Bath applications of structurally dissimilar anaesthetics, isoflurane - a halogenated ether, and Althesin - a steroidal preparation, in concentrations of 0.5-2.5 minimum alveolar concentration (MAC) and 10-1300 μM, respectively, produced a small hyperpolarization (3-5 mV) which was associated with an increase in input conductance (10-30%). The lower concentrations (0.5-1.5 MAC and 10-200 μM) of these agents which are most relevant to the production of unconsciousness did not significantly affect the passive membrane properties. However, they produced striking decreases in spontaneous activities and the repetitive spike firing evoked by orthodromic (electrical) stimulation or intracellular current injections. Because the observed changes in membrane properties could not explain the reduction in neuronal excitability, the effects of anaesthetics were investigated extensively on excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs). The application of isoflurane or Althesin induced a dose-dependent, reversible depression in the amplitude of EPSPs, with EC₅₀s of 1 MAC and ~50 μM, respectively. The IPSPs also were reduced in a dose-dependent manner. In order to eliminate possible shunting of the EPSPs by the GABA-activated Cl-conductance that produces the IPSP in the observed EPSP-IPSP sequence, a GABA[symbol omitted]-antagonist, bicuculline, was additionally applied. Despite this IPSP-blockade, the anaesthetics strongly depressed the EPSPs as well as epileptiform activities evoked by subpial electrical stimulation. In cognizance of the possibility that a postsynaptic attenuation of responsiveness to transmitter substances may be involved in the EPSP depression, the neuronal sensitivities to acetylcholine, glutamate. and GABA were determined. Anaesthetic administration markedly reduced the depolarizations and associated conductance changes evoked by dendritic applications of acetylcholine, glutamate and N-methyl-D-aspartate (NMDA). The hyperpolarizing responses to somatic applications of GABA were not affected significantly whereas the depolarizing effects observed with its dendritic application were slightly depressed. Same degree of selectivity also was evident from the lower EC₅₀s for the isoflurane- and Althesin-induced depressions of responses to acetylcholine compared with glutamate. Under in vitro conditions of hypomagnesia the responses to acetylcholine were totally blocked and the order of depression in the responses to GABA and glutamate was reversed; this may be of importance in the mechanism for the known increase in anaesthetic requirements in clinical syndromes associated with hypomagnesaemia. Because the genesis of synaptic transients is affected by Ca²⁺ influx or disposition, the interactions of anaesthetics were investigated on spike afterhyperpolarizations (AHPs). The AHPs which are produced specifically by a Ca²⁺ -activated K⁺ -conductance were suppressed by the anaesthetics in a dose-dependent manner under conditions where contaminating IPSPs had been blocked by bicuculline. Since the passive membrane properties were unaffected, an interference with a transmembrane Ca⁺ -influx may be involved in the anaesthetic actions. The effects of anaesthetics on glutamate-induced and voltage-dependent increases in intraneuronal Ca²⁺ ([Ca²⁺]i) were determined in cultured hippocampal neurons with a Ca-sensitive probe (Fura-2) and microspectro- fluorometric techniques. Isoflurane application depressed the increases in [Ca²⁺]i. produced by application of glutamate under conditions where its actions would be favoured at NMDA- and quisqualate-subtypes of receptors. K⁺ -induced increases in [Ca²⁺]i also were reduced by application of isoflurane, probably due to actions on voltage-dependent Ca-channels in the membrane. These investigations have provided evidence for the first time that excitatory transmitter actions in neocortex are selectively depressed by anaesthesia. A plausible mechanism would include suppression of the postsynaptic Ca-conductances associated with the AHPs and glutamatergic, as well as cholinergic interactions at pre- and post-synaptic sites on neurons involved in neocortical arousal.Medicine, Faculty ofAnesthesiology, Pharmacology and Therapeutics, Department ofGraduat

PSF engineering in multifocus microscopy for increased depth volumetric imaging

International audienceImaging and localizing single molecules with high accuracy in a 3D volume is a challenging task. Here we combine multifocal microscopy, a recently developed volumetric imaging technique, with point spread function engineering to achieve an increased depth for single molecule imaging. Applications in 3D single molecule localization-based super-resolution imaging is shown over an axial depth of 4 µm as well as for the tracking of diffusing beads in a fluid environment over 8 µm