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Anuran saxiphilins (Sxphs) are "toxin sponge" proteins thought to prevent the lethal effects of small-molecule neurotoxins through sequestration. Here, we present a protocol for the expression, purification, and characterization of Sxphs. We describe steps for using thermofluor, fluorescence polarization, and isothermal titration calorimetry assays that probe Sxph:saxitoxin interactions using a range of sample quantities. These assays are generalizable and can be used for other paralytic shellfish poisoning toxin-binding proteins. For complete details on the use and execution of this protocol, please refer to Chen et al. (2022). 1 ., Competing Interests: Declaration of interests J.D.B. is a cofounder and holds equity shares in SiteOne Therapeutics, Inc., a start-up company interested in developing subtype-selective modulators of sodium channels. Patent applications have been filed for this work., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

American bullfrog ( Rana castesbeiana ) saxiphilin ( Rc Sxph) is a high-affinity "toxin sponge" protein thought to prevent intoxication by saxitoxin (STX), a lethal bis-guanidinium neurotoxin that causes paralytic shellfish poisoning (PSP) by blocking voltage-gated sodium channels (Na V s). How specific Rc Sxph interactions contribute to STX binding has not been defined and whether other organisms have similar proteins is unclear. Here, we use mutagenesis, ligand binding, and structural studies to define the energetic basis of Sxph:STX recognition. The resultant STX "recognition code" enabled engineering of Rc Sxph to improve its ability to rescue Na V s from STX and facilitated discovery of 10 new frog and toad Sxphs. Definition of the STX binding code and Sxph family expansion among diverse anurans separated by ∼140 My of evolution provides a molecular basis for understanding the roles of toxin sponge proteins in toxin resistance and for developing novel proteins to sense or neutralize STX and related PSP toxins.

Multidrug and toxic compound extrusion (MATE) transporters mediate excretion of xenobiotics and toxic metabolites, thereby conferring multidrug resistance in bacterial pathogens and cancer cells. Structural information on the alternate conformational states and knowledge of the detailed mechanism of MATE transport are of great importance for drug development. However, the structures of MATE transporters are only known in V-shaped outward-facing conformations. Here, we present the crystal structure of a MATE transporter from Pyrococcus furiosus (PfMATE) in the long-sought-after inward-facing state, which was obtained after crystallization in the presence of native lipids. Transition from the outward-facing state to the inward-facing state involves rigid body movements of transmembrane helices (TMs) 2-6 and 8-12 to form an inverted V, facilitated by a loose binding of TM1 and TM7 to their respective bundles and their conformational flexibility. The inward-facing structure of PfMATE in combination with the outward-facing one supports an alternating access mechanism for the MATE family transporters., Competing Interests: The authors declare no conflict of interest.

Background: Although the negative health consequences of the exposure to second hand tobacco smoke during childhood are already known, evidence on the economic consequences is still rare. The aim of this study was to estimate excess healthcare costs of exposure to tobacco smoke in German children., Methods: The study is based on data from two birth cohort studies of 3,518 children aged 9-11 years with information on healthcare utilisation and tobacco smoke exposure: the GINIplus study (German Infant Study On The Influence Of Nutrition Intervention Plus Environmental And Genetic Influences On Allergy Development) and the LISAplus study (Influence of Life-Style Factors On The Development Of The Immune System And Allergies In East And West Germany Plus The Influence Of Traffic Emissions And Genetics). Direct medical costs were estimated using a bottom-up approach (base year 2007). We investigated the impact of tobacco smoke exposure in different environments on the main components of direct healthcare costs using descriptive analysis and a multivariate two-step regression analysis., Results: Descriptive analysis showed that average annual medical costs (physician visits, physical therapy and hospital treatment) were considerably higher for children exposed to second-hand tobacco smoke at home (indoors or on patio/balcony) compared with those who were not exposed. Regression analysis confirmed these descriptive trends: the odds of positive costs and the amount of total costs are significantly elevated for children exposed to tobacco smoke at home after adjusting for confounding variables. Combining the two steps of the regression model shows smoking attributable total costs per child exposed at home of €87 [10-165] (patio/balcony) and €144 [6-305] (indoors) compared to those with no exposure. Children not exposed at home but in other places showed only a small, but not significant, difference in total costs compared to those with no exposure., Conclusions: This study shows adverse economic consequences of second-hand smoke in children depending on proximity of exposure. Tobacco smoke exposure seems to affect healthcare utilisation in children who are not only exposed to smoke indoors but also if parents reported exclusively smoking on patio or balcony. Preventing children from exposure to second-hand tobacco smoke might thus be desirable not only from a health but also from an economic perspective.