Single-molecule dynamics of surface lipoproteins in bacteroides indicate similarities and cooperativity.

The gut microbiota comprises hundreds of species with a composition shaped by the available glycans. The well-studied starch utilization system (Sus) is a prototype for glycan uptake in the human gut bacterium Bacteroides thetaiotaomicron (Bt). Each Sus-like system includes outer-membrane proteins, which translocate glycan into the periplasm, and one or more cell-surface glycoside hydrolases, which break down a specific (cognate) polymer substrate. Although the molecular mechanisms of the Sus system are known, how the Sus and Sus-like proteins cooperate remains elusive. Previously, we used single-molecule and super-resolution fluorescence microscopy to show that SusG is mobile on the outer membrane and slows down in the presence of starch. Here, we compare the dynamics of three glycoside hydrolases: SusG, Bt4668, and Bt1760, which target starch, galactan, and levan, respectively. We characterized the diffusion of each surface hydrolase in the presence of its cognate glycan and found that all three enzymes are mostly immobile in the presence of the polysaccharide, consistent with carbohydrate binding. Moreover, experiments in glucose versus oligosaccharides suggest that the enzyme dynamics depend on their expression level. Furthermore, we characterized enzyme diffusion in a mixture of glycans and found that noncognate polysaccharides modify the dynamics of SusG and Bt1760 but not Bt4668. We investigated these systems with polysaccharide mixtures and genetic knockouts and found that noncognate polysaccharides modify hydrolase dynamics through some combination of nonspecific protein interactions and downregulation of the hydrolase. Overall, these experiments extend our understanding of how Sus-like lipoprotein dynamics can be modified by changing carbohydrate conditions and the expression level of the enzyme.

Force measurements show that uL4 and uL24 mechanically stabilize a fragment of 23S rRNA essential for ribosome assembly.

In vitro reconstitution studies have shown that ribosome assembly is highly cooperative and starts with the binding of a few ribosomal (r-) proteins to rRNA. It is unknown how these early binders act. Focusing on the initial stage of the assembly of the large subunit of the Escherichia coli ribosome, we prepared a 79-nucleotide-long region of 23S rRNA encompassing the binding sites of the early binders uL4 and uL24. Force signals were measured in a DNA/RNA dumbbell configuration with a double optical tweezers setup. The rRNA fragment was stretched until unfolded, in the absence or in the presence of the r-proteins (either uL4, uL24, or both). We show that the r-proteins uL4 and uL24 individually stabilize the rRNA fragment, both acting as molecular clamps. Interestingly, this mechanical stabilization is enhanced when both proteins are bound simultaneously. Independently, we observe a cooperative binding of uL4 and uL24 to the rRNA fragment. These two aspects of r-proteins binding both contribute to the efficient stabilization of the 3D structure of the rRNA fragment under investigation. We finally consider implications of our results for large ribosomal subunit assembly.

[HIV infection and social condition: a disease which remains singular]. / Infection par le VIH et société: une maladie qui demeure singulière.

Increased efficacy of HIV therapy has resulted in a dramatic improvement in patient's health condition regarding life expectancy and life quality. However, such favorable evolution had no significant impact on the social condition of the population living with HIV, which remains more exposed to socio-economical difficulties and to different forms of stigmatization and discrimination. Public policies do address these issues. The national HIV/AIDS and STI strategic plan 2010-2014 provides a range of actions aimed at fighting against discriminations and improving social care for the most vulnerable people living with HIV. It notably tries to achieve equal access to programs and services developed for patients with chronic diseases and to improve their condition regarding access to care, employment, housing and income. Implementation of these measures, however, proves difficult. Addressing social and societal concerns that HIV infection still raises is part of the care and support that should be offered to patients. Providing this comprehensive approach remains critical for ensuring optimized individual therapeutic outcomes as well as an efficient collective response to the epidemic.

NOBIAS: Analyzing anomalous diffusion in single-molecule tracks with nonparametric Bayesian inference.

Single particle tracking (SPT) enables the investigation of biomolecular dynamics at a high temporal and spatial resolution in living cells, and the analysis of these SPT datasets can reveal biochemical interactions and mechanisms. Still, how to make the best use of these tracking data for a broad set of experimental conditions remains an analysis challenge in the field. Here, we develop a new SPT analysis framework: NOBIAS (NOnparametric Bayesian Inference for Anomalous Diffusion in Single-Molecule Tracking), which applies nonparametric Bayesian statistics and deep learning approaches to thoroughly analyze SPT datasets. In particular, NOBIAS handles complicated live-cell SPT data for which: the number of diffusive states is unknown, mixtures of different diffusive populations may exist within single trajectories, symmetry cannot be assumed between the x and y directions, and anomalous diffusion is possible. NOBIAS provides the number of diffusive states without manual supervision, it quantifies the dynamics and relative populations of each diffusive state, it provides the transition probabilities between states, and it assesses the anomalous diffusion behavior for each state. We validate the performance of NOBIAS with simulated datasets and apply it to the diffusion of single outer-membrane proteins in Bacteroides thetaiotaomicron. Furthermore, we compare NOBIAS with other SPT analysis methods and find that, in addition to these advantages, NOBIAS is robust and has high computational efficiency and is particularly advantageous due to its ability to treat experimental trajectories with asymmetry and anomalous diffusion.

RNA Folding and Unfolding Under Force: Single-Molecule Experiments and Their Analysis.

We have previously described (Geffroy et al. Methods Mol Biol 1665:25-40, 2018) how to unfold (or fold) a single RNA molecule under force using a dual-beam optical trap setup. In this chapter, we complementarily describe how to analyze the corresponding data and how to interpret it in terms of RNA three-dimensional structure. As with all single-molecule methods, single RNA molecule force data often exhibit several discrete states where state-to-state transitions are blurred in a noisy signal. In order to cope with this limitation, we have implemented a novel strategy to analyze the data, which uses a hidden Markov modeling procedure. A representative example of such an analysis is presented.

RNA Unzipping and Force Measurements with a Dual Optical Trap.

In order to mechanically unfold a single RNA molecule, an RNA/DNA hybrid construction is prepared which allows specific attachment to two micrometer-sized beads. A dual-beam optical trap thus holding the construct in solution captures the beads separately. Unfolding of a molecule is obtained by increasing the distance between the traps, one trap being slowly moved while the other is held fixed. Force is measured to sub-piconewton precision by back focal plane interferometry of the bead in the fixed trap. The experiment allows us to measure structure and base-sequence-dependent force signals. In this chapter, important technical aspects of this type of single-molecule force measurements are considered.