Induction of Internal Circadian Desynchrony by Misaligning Zeitgebers

24-hour rhythms in physiology and behaviour are orchestrated by an endogenous circadian clock system. In mammals, these clocks are hierarchically organized with a master pacemaker residing in the hypothalamic suprachiasmatic nucleus (SCN). External time signal – so-called zeitgebers – align internal with geophysical time. During shift work, zeitgeber input conflicting with internal time induces circadian desynchrony which, in turn, promotes metabolic and psychiatric disorders. However, little is known about how internal desynchrony is expressed at the molecular level under chronodisruptive environmental conditions. We here investigated the effects of zeitgeber misalignment on circadian molecular organisation by combining 28-hour light-dark (LD-28) cycles with either 24-hour (FF-24) or 28-hour feeding-fasting (FF-28) regimes in mice. We found that FF cycles showed strong effects on peripheral clocks, while having little effect on centrally coordinated activity rhythms. Systemic, i.e., across-tissue internal circadian desynchrony was induced within four days in LD-28/FF-24, while phase coherence between tissue clocks was largely maintained under LD-28/FF-28 conditions. In contrast, temporal coordination of clock gene activity across tissues was reduced under LD-28/FF-28 conditions compared to LD-28/FF-24. These results indicate that timed food intake may improve internal synchrony under disruptive zeitgeber conditions but may, at the same time, weaken clock function at the tissue level.

Rheological and structural properties of lecithin-based organogels

The objectives of this study were to develop lecithin-based organogels made of a biocompatible organic phase and to study the structural and rheological properties of organogels made with different organic phases. The materials used were soybean lecithin of >90% purity (phosphatidylcholine), distilled water and the following organic phases: isooctane (ISO), mineral oil (MO) and isopropylpalmitate (IPP). Phase diagrams for the PC/Water/ISO, PC/Water/ISO/MO (20:80 vo1/vo1), PC/Water/IPP,PC/Water/IPP/MO (50:50vo1/vo1 and 30:70vo1/vo1) and Pc/Water/MO systems were analysed using polarised light microscopy. A narrow region of organogel formation was observed in all systems at molar ratios of water to lecithin between values of 0.4 and 4, depending on the organic phase used. Small-angle X-ray diffraction studies for the PC/Water/ISO system revealed the existence of a highly ordered two-dimensional crystal lattice likely formed by the bundling of the cylindrical reverse micellar tubes. The results for the Pc/Water/IPP and PC/Water/MO systems indicated that there was not enough structural material in the organogels to observe any level of molecular organisation. In summary, this study demonstrated that it was possible to develop biocompatible lecithin-based organogels.

Rheological and structural properties of lecithin-based organogels

The objectives of this study were to develop lecithin-based organogels made of a biocompatible organic phase and to study the structural and rheological properties of organogels made with different organic phases. The materials used were soybean lecithin of >90% purity (phosphatidylcholine), distilled water and the following organic phases: isooctane (ISO), mineral oil (MO) and isopropylpalmitate (IPP). Phase diagrams for the PC/Water/ISO, PC/Water/ISO/MO (20:80 vo1/vo1), PC/Water/IPP,PC/Water/IPP/MO (50:50vo1/vo1 and 30:70vo1/vo1) and Pc/Water/MO systems were analysed using polarised light microscopy. A narrow region of organogel formation was observed in all systems at molar ratios of water to lecithin between values of 0.4 and 4, depending on the organic phase used. Small-angle X-ray diffraction studies for the PC/Water/ISO system revealed the existence of a highly ordered two-dimensional crystal lattice likely formed by the bundling of the cylindrical reverse micellar tubes. The results for the Pc/Water/IPP and PC/Water/MO systems indicated that there was not enough structural material in the organogels to observe any level of molecular organisation. In summary, this study demonstrated that it was possible to develop biocompatible lecithin-based organogels.

Biofunctionality with a twist: the importance of molecular organisation, handedness and configuration in synthetic biomaterial design

This review highlights the importance of incorporating molecular organisation, spatial configuration and handedness in biomaterial design to arrive at improved native biomolecule interactions.

Stimulation strength controls the rate of initiation but not the molecular organisation of TCR-induced signalling

Millions of naïve T cells with different TCRs may interact with a peptide-MHC ligand, but very few will activate. Remarkably, this fine control is orchestrated using a limited set of intracellular machinery. It remains unclear whether changes in stimulation strength alter the programme of signalling events leading to T cell activation. Using mass cytometry to simultaneously measure multiple signalling pathways during activation of murine CD8+ T cells, we found a programme of distal signalling events that is shared, regardless of the strength of TCR stimulation. Moreover, the relationship between transcription of early response genes Nr4a1 and Irf8 and activation of the ribosomal protein S6 is also conserved across stimuli. Instead, we found that stimulation strength dictates the rate with which cells initiate signalling through this network. These data suggest that TCR-induced signalling results in a coordinated activation program, modulated in rate but not organization by stimulation strength.

Binding partners regulate unfolding of myosin VI to activate the molecular motor

ABSTRACTMyosin VI is the only minus-end actin motor and is coupled to various cellular processes ranging from endocytosis to transcription. This multi-potent nature is achieved through alternative isoform splicing and interactions with a network of binding partners. How the regulation mechanism of myosin VI varies between different isoforms and binding partners remains unexplored. Here, we have compared the regulation of two myosin VI splice isoforms by two different binding partners. By combining biochemical and single-molecule approaches, we propose that myosin VI regulation follows a generic mechanism, independently of the spliced isoform and the binding partner involved. We describe how myosin VI adopts an autoinhibited backfolded state which is unfolded by binding partners. This unfolding activates the motor and can subsequently trigger dimerization. We have further expanded our study by using live single molecule imaging to investigate the impact of binding partners upon myosin VI molecular organisation and dynamics. Overall, binding partners determine myosin VI function.

Central role and structure of the membrane pseudokinase YukC in the antibacterial Bacillus subtilis Type VIIb Secretion System

Type VIIb Secretion System (T7SSb) has been recently identified in Firmicutes resembling the mycobacterial T7SSa. Despite limited sequence homology, T7SSa and T7SSb have substrates with striking structural similarities, the WXG100 proteins. Recent advances in Staphylococcus spp. proposed that T7SSb is involved in intra-species competition. However, the architecture and mechanism of action of this secretion complex remain largely obscure. Here, we investigate the T7SSb of Bacillus subtilis as a model system. We report the first evidence of B. subtilis ability to mediate intra- and inter-species antibacterial activity in a T7SSb-dependent manner. Then, we present the first systematic investigation of the T7SSb protein-protein network, revealing novel interactions and highlighting the central role of the pseudokinase subunit YukC in the assembly of the system. Its direct interaction with a T7SSb-secreted toxin supports its role in recruiting substrates to the secretion machinery. Finally, we solved the crystal structure of full-length transmembrane YukC defining novel structural motifs and suggesting that intrinsic flexibility modulates the orientation of the pseudokinase domains and YukC function. Overall, our results provide a better understanding on the role and molecular organisation of the T7SSb, opening new perspectives for the comprehension of this poorly characterized molecular machine.