SWEETs are transporters with homologs in Archeae, plants, some fungi, and animals. As the only transporters known to facilitate the cellular release of sugars in plants, SWEETs play critical roles in the allocation of sugars from photosynthetic leaves to storage tissues in seeds, fruits, and tubers. Here, we report the design and use of genetically encoded biosensors to measure the activity of SWEETs. We created a SweetTrac1 sensor by inserting a circularly permutated green fluorescent protein into the Arabidopsis SWEET1, resulting in a chimera that translates substrate binding during the transport cycle into detectable changes in fluorescence intensity. We demonstrate that a combination of cell sorting and bioinformatics can accelerate the design of biosensors and formulate a mass action kinetics model to correlate the fluorescence response of SweetTrac1 with the transport of glucose. Our analysis suggests that SWEETs are low-affinity, symmetric transporters that can rapidly equilibrate intra- and extracellular concentrations of sugars. This approach can be extended to SWEET homologs and other transporters.
SummaryMechanistic understanding of the impacts of the gut microbiota on human health has been hampered by limited throughput in animal models. To enable systematic interrogation of gut-relevant microbial communities, here we generated hundreds of in vitro communities cultured from diverse stool samples in various media. Species composition revealed stool-derived communities that are phylogenetically complex, diverse, stable, and highly reproducible. Community membership depended on both medium and initial inoculum, with certain media preserving inoculum compositions. Different inocula yielded different community compositions, indicating their potential for personalized therapeutics. Communities were robust to freezing and large-volume culturing, enabling future translational applications. Defined communities were generated from isolates and reconstituted growth and composition similar to those of communities derived from stool inocula. Finally, in vitro experiments probing the response to ciprofloxacin successfully predicted many changes observed in vivo, including the resilience and sensitivity of each Bacteroides species. Thus, stool-derived in vitro communities constitute a powerful resource for microbiota research.
The neuromuscular junction (NMJ) is a specialized cholinergic synaptic interface between a motor neuron and a skeletal muscle fiber that translates presynaptic electrical impulses into motor function. NMJ formation and maintenance require tightly regulated signaling and cellular communication among motor neurons, myogenic cells, and Schwann cells. Neuromuscular diseases (NMDs) can result in loss of NMJ function and motor input leading to paralysis or even death. Although small animal models have been instrumental in advancing our understanding of the NMJ structure and function, the complexities of studying this multi-tissue system in vivo and poor clinical outcomes of candidate therapies developed in small animal models has driven the need for in vitro models of functional human NMJ to complement animal studies. In this review, we discuss prevailing models of NMDs and highlight the current progress and ongoing challenges in developing human iPSC-derived (hiPSC) 3D cell culture models of functional NMJs. We first review in vivo development of motor neurons, skeletal muscle, Schwann cells, and the NMJ alongside current methods for directing the differentiation of relevant cell types from hiPSCs. We further compare the efficacy of modeling NMDs in animals and human cell culture systems in the context of five NMDs: amyotrophic lateral sclerosis, myasthenia gravis, Duchenne muscular dystrophy, myotonic dystrophy, and Pompe disease. Finally, we discuss further work necessary for hiPSC-derived NMJ models to function as effective personalized NMD platforms.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.