The substrate import mechanism of the human serotonin transporter.

The serotonin transporter (SERT) initiates the reuptake of extracellular serotonin in the synapse to terminate neurotransmission. The cryogenic electron microscopy structures of SERT bound to ibogaine and the physiological substrate serotonin resolved in different states have provided a glimpse of the functional conformations at atomistic resolution. However, the conformational dynamics and structural transitions to intermediate states are not fully understood. Furthermore, the molecular basis of how serotonin is recognized and transported remains unclear. In this study, we performed unbiased microsecond-long simulations of the human SERT to investigate the structural dynamics to various intermediate states and elucidated the complete substrate import pathway. Using Markov state models, we characterized a sequential order of conformational-driven ion-coupled substrate binding and transport events and calculated the free energy barriers of conformation transitions associated with the import mechanism. We find that the transition from the occluded to inward-facing state is the rate-limiting step for substrate import and that the substrate decreases the free energy barriers to achieve the inward-facing state. Our study provides insights on the molecular basis of dynamics-driven ion-substrate recognition and transport of SERT that can serve as a model for other closely related neurotransmitter transporters.

Dietary assessment in UK Biobank: an evaluation of the performance of the touchscreen dietary questionnaire.

UK Biobank is an open access prospective cohort of 500 000 men and women. Information on the frequency of consumption of main foods was collected at recruitment with a touchscreen questionnaire; prior to examining the associations between diet and disease, it is essential to evaluate the performance of the dietary touchscreen questionnaire. The objectives of the present paper are to: describe the repeatability of the touchscreen questionnaire in participants (n 20 348) who repeated the assessment centre visit approximately 4 years after recruitment, and compare the dietary touchscreen variables with mean intakes from participants (n 140 080) who completed at least one of the four web-based 24-h dietary assessments post-recruitment. For fish and meat items, 90 % or more of participants reported the same or adjacent category of intake at the repeat assessment visit; for vegetables and fruit, and for a derived partial fibre score (in fifths), 70 % or more of participants were classified into the same or adjacent category of intake (κweighted > 0·50 for all). Participants were also categorised based on their responses to the dietary touchscreen questionnaire at recruitment, and within each category the group mean intake of the same food group or nutrient from participants who had completed at least one web-based 24-h dietary assessment was calculated. The comparison showed that the dietary touchscreen variables, available on the full cohort, reliably rank participants according to intakes of the main food groups.

Murine macrophages produce endothelin-1 after microbial stimulation.

Endothelin-1 (ET-1) was originally characterized as a potent vasoconstrictor secreted by the endothelium and participating in the regulation of vascular tone. Subsequent analysis has revealed ET-1 to be a multifunctional peptide produced by a wide variety of cells and tissues under normal and pathologic conditions. The importance of macrophages as a source of ET-1 during infection and inflammation is supported by clinical observations in humans and in animal models of inflammation. We hypothesize that the production of ET-1 is part of the characteristic macrophage response to infection, and have begun to investigate the ability of various classes of microbes or microbial products to induce macrophage ET-1 production. We report the production of ET-1 by murine macrophages in response to stimulation with both gram-positive and gram-negative bacteria. Stimulation of macrophages with yeast (Candida albicans or Saccharomyces cerevisiae) or the protozoan parasite Leishmania major, elicited no significant release of ET-1. The production of ET-1 in response to lipopolysaccharide (LPS) was dose and time dependent, and required the expression of a functional toll-like receptor 4 (TLR4). Pharmacologic inhibition of the transcription factor, nuclear factor-kappaB (NF-kappaB) suppressed LPS-induced ET-1 production. Our findings complement the growing body of literature implicating a role for macrophage-derived ET-1 in inflammatory pathologies. The production of ET-1 by macrophages during infection and inflammation has the potential to affect tissue perfusion, leukocyte extravasation, and immune cell function.