The tarantula toxin GxTx detains K+ channel gating charges in their resting conformation.

Allosteric ligands modulate protein activity by altering the energy landscape of conformational space in ligand-protein complexes. Here we investigate how ligand binding to a K+ channel's voltage sensor allosterically modulates opening of its K+-conductive pore. The tarantula venom peptide guangxitoxin-1E (GxTx) binds to the voltage sensors of the rat voltage-gated K+ (Kv) channel Kv2.1 and acts as a partial inverse agonist. When bound to GxTx, Kv2.1 activates more slowly, deactivates more rapidly, and requires more positive voltage to reach the same K+-conductance as the unbound channel. Further, activation kinetics are more sigmoidal, indicating that multiple conformational changes coupled to opening are modulated. Single-channel current amplitudes reveal that each channel opens to full conductance when GxTx is bound. Inhibition of Kv2.1 channels by GxTx results from decreased open probability due to increased occurrence of long-lived closed states; the time constant of the final pore opening step itself is not impacted by GxTx. When intracellular potential is less than 0 mV, GxTx traps the gating charges on Kv2.1's voltage sensors in their most intracellular position. Gating charges translocate at positive voltages, however, indicating that GxTx stabilizes the most intracellular conformation of the voltage sensors (their resting conformation). Kinetic modeling suggests a modulatory mechanism: GxTx reduces the probability of voltage sensors activating, giving the pore opening step less frequent opportunities to occur. This mechanism results in K+-conductance activation kinetics that are voltage-dependent, even if pore opening (the rate-limiting step) has no inherent voltage dependence. We conclude that GxTx stabilizes voltage sensors in a resting conformation, and inhibits K+ currents by limiting opportunities for the channel pore to open, but has little, if any, direct effect on the microscopic kinetics of pore opening. The impact of GxTx on channel gating suggests that Kv2.1's pore opening step does not involve movement of its voltage sensors

Microbial community function and bacterial pathogen composition in pit latrines in peri-urban Malaw

Despite the widespread global reliance on pit latrines as improved sanitation systems, the decomposition of waste within pit latrines is poorly understood. One area needing elucidation is the characterization and function of microbial communities within pit latrines. To address this gap, we characterized the microbial communities of 55 lined pit latrines at three sampling layers from two communities in peri-urban Malawi. The microbial communities of the fecal sludge samples were analyzed for beta diversity, pathogen presence, and functional profiling. Household surveys were conducted and used to compare microbial community patterns to household characteristics and pit latrine use patterns. Compared to activated sludge, anaerobic digestion in municipal wastewater systems, and human gut microbiomes, pit latrines were found to contain unique microbial communities. While the microbial community composition as a whole did not vary by sampling depth, pathogen composition varied by sampling depth, location, and household water source. The inferred microbial function also varied by depth (e.g., increase in methanogens and decrease in aerobes with depth). The richness of lined pit latrines determined from surface samples from eight latrines was found to be representative for a given area. Samples from middle and lower depths collected using a Gulper pump did not provide more information on richness, a result that informs future sampling designs. These findings are important for improving waste-based epidemiology (WBE) approaches to understand community health and waste degradation characterization of lined pit latrines

Impact of international travel and diarrhea on gut microbiome and resistome dynamics

International travel contributes to the global spread of antimicrobial resistance. Travelers\u27 diarrhea exacerbates the risk of acquiring multidrug-resistant organisms and can lead to persistent gastrointestinal disturbance post-travel. However, little is known about the impact of diarrhea on travelers\u27 gut microbiomes, and the dynamics of these changes throughout travel. Here, we assembled a cohort of 159 international students visiting the Andean city of Cusco, Peru and applied next-generation sequencing techniques to 718 longitudinally-collected stool samples. We find that gut microbiome composition changed significantly throughout travel, but taxonomic diversity remained stable. However, diarrhea disrupted this stability and resulted in an increased abundance of antimicrobial resistance genes that can remain high for weeks. We also identified taxa differentially abundant between diarrheal and non-diarrheal samples, which were used to develop a classification model that distinguishes between these disease states. Additionally, we sequenced the genomes of 212 diarrheagenic Escherichia coli isolates and found those from travelers who experienced diarrhea encoded more antimicrobial resistance genes than those who did not. In this work, we find the gut microbiomes of international travelers\u27 are resilient to dysbiosis; however, they are also susceptible to colonization by multidrug-resistant bacteria, a risk that is more pronounced in travelers with diarrhea

Microbial community function and bacterial pathogen composition in pit latrines in peri-urban Malawi

Despite the widespread global reliance on pit latrines as improved sanitation systems, the decomposition of waste within pit latrines is poorly understood. One area needing elucidation is the characterization and function of microbial communities within pit latrines. To address this gap, we characterized the microbial communities of 55 lined pit latrines at three sampling layers from two communities in peri-urban Malawi. The microbial communities of the fecal sludge samples were analyzed for beta diversity, pathogen presence, and functional profiling. Household surveys were conducted and used to compare microbial community patterns to household characteristics and pit latrine use patterns. Compared to activated sludge, anaerobic digestion in municipal wastewater systems, and human gut microbiomes, pit latrines were found to contain unique microbial communities. While the microbial community composition as a whole did not vary by sampling depth, pathogen composition varied by sampling depth, location, and household water source. The inferred microbial function also varied by depth (e.g., increase in methanogens and decrease in aerobes with depth). The richness of lined pit latrines determined from surface samples from eight latrines was found to be representative for a given area. Samples from middle and lower depths collected using a Gulper pump did not provide more information on richness, a result that informs future sampling designs. These findings are important for improving waste-based epidemiology (WBE) approaches to understand community health and waste degradation characterization of lined pit latrines

Azide-Alkyne Click Conjugation on Quantum Dots by Selective Copper Coordination.

Functionalization of nanocrystals is essential for their practical application, but synthesis on nanocrystal surfaces is limited by incompatibilities with certain key reagents. The copper-catalyzed azide-alkyne cycloaddition is among the most useful methods for ligating molecules to surfaces, but has been largely useless for semiconductor quantum dots (QDs) because Cu+ ions quickly and irreversibly quench QD fluorescence. To discover nonquenching synthetic conditions for Cu-catalyzed click reactions on QD surfaces, we developed a combinatorial fluorescence assay to screen >2000 reaction conditions to maximize cycloaddition efficiency while minimizing QD quenching. We identify conditions for complete coupling without significant quenching, which are compatible with common QD polymer surfaces and various azide/alkyne pairs. Based on insight from the combinatorial screen and mechanistic studies of Cu coordination and quenching, we find that superstoichiometric concentrations of Cu can promote full coupling if accompanied by ligands that selectively compete with the Cu from the QD surface but allow it to remain catalytically active. Applied to the conjugation of a K+ channel-specific peptidyl toxin to CdSe/ZnS QDs, we synthesize unquenched QD conjugates and image their specific and voltage-dependent affinity for K+ channels in live cells