Acrylic-based culture plate format perfusion device to establish liver endothelial-epithelial interface.

Microphysiological Systems (MPSs) or organs-on-chips, are microfluidic devices used to model human physiology in vitro. Polydimethylsiloxane (PDMS) is the most widely used material for organs-on-chips due to its established fabrication methods and biocompatibility properties. However, non-specific binding of small molecules limits PDMS for drug screening applications. Here, we designed a novel acrylic-based MPS to capture the physiological architecture that is observed universally in tissues across the body: the endothelial-epithelial interface (EEI). To reconstruct the EEI biology, we designed a membrane-based chip that features endothelial cells on the underside of the membrane exposed to mechanical shear from the path of media flow, and epithelial cells on the opposite side of the membrane protected from flow, as they are in vivo. We used a liver model with a hepatic progenitor cell line and human umbilical vein endothelial cells to assess the biological efficacy of the MPS. We computationally modeled the physics that govern the function of perfusion through the MPS. Empirically, efficacy was measured by comparing differentiation of the hepatic progenitor cells between the MPS and 2D culture conditions. We demonstrated that the MPS significantly improved hepatocyte differentiation, increased extracellular protein transport, and raised hepatocyte sensitivity to drug treatment. Our results strongly suggest that physiological perfusion has a profound effect on proper hepatocyte function, and the modular chip design motivates opportunities for future study of multi-organ interactions.

Dominant negative effects by inactive Spa47 mutants inhibit T3SS function and Shigella virulence.

Type three secretion systems (T3SS) are complex nano-machines that evolved to inject bacterial effector proteins directly into the cytoplasm of eukaryotic cells. Many high-priority human pathogens rely on one or more T3SSs to cause disease and evade host immune responses, underscoring the need to better understand the mechanisms through which T3SSs function and their role(s) in supporting pathogen virulence. We recently identified the Shigella protein Spa47 as an oligomerization-activated T3SS ATPase that fuels the T3SS and supports overall Shigella virulence. Here, we provide both in vitro and in vivo characterization of Spa47 oligomerization and activation in the presence and absence of engineered ATPase-inactive Spa47 mutants. The findings describe mechanistic details of Spa47-catalyzed ATP hydrolysis and uncover critical distinctions between oligomerization mechanisms capable of supporting ATP hydrolysis in vitro and those that support T3SS function in vivo. Concentration-dependent ATPase kinetics and experiments combining wild-type and engineered ATPase inactive Spa47 mutants found that monomeric Spa47 species isolated from recombinant preparations exhibit low-level ATPase activity by forming short-lived oligomers with active site contributions from at least two protomers. In contrast, isolated Spa47 oligomers exhibit enhanced ATP hydrolysis rates that likely result from multiple preformed active sites within the oligomeric complex, as is predicted to occur within the context of the type three secretion system injectisome. High-resolution fluorescence microscopy, T3SS activity, and virulence phenotype analyses of Shigella strains co-expressing wild-type Spa47 and the ATPase inactive Spa47 mutants demonstrate that the N-terminus of Spa47, not ATPase activity, is responsible for incorporation into the injectisome where the mutant strains exhibit a dominant negative effect on T3SS function and Shigella virulence. Together, the findings presented here help to close a significant gap in our understanding of how T3SS ATPases are activated and define restraints with respect to how ATP hydrolysis is ultimately coupled to T3SS function in vivo.

Visible-Light-Driven Valorization of Biomass Intermediates Integrated with H2 Production Catalyzed by Ultrathin Ni/CdS Nanosheets.

Photocatalytic upgrading of crucial biomass-derived intermediate chemicals (i.e., furfural alcohol, 5-hydroxymethylfurfural (HMF)) to value-added products (aldehydes and acids) was carried out on ultrathin CdS nanosheets (thickness ∼1 nm) decorated with nickel (Ni/CdS). More importantly, simultaneous H2 production was realized upon visible light irradiation under ambient conditions utilizing these biomass intermediates as proton sources. The remarkable difference in the rates of transformation of furfural alcohol and HMF to their corresponding aldehydes in neutral water was observed and investigated. Aided by theoretical computation, it was rationalized that the slightly stronger binding affinity of the aldehyde group in HMF to Ni/CdS resulted in the lower transformation of HMF to 2,5-diformylfuran compared to that of furfural alcohol to furfural. Nevertheless, photocatalytic oxidation of furfural alcohol and HMF under alkaline conditions led to complete transformation to the respective carboxylates with concomitant production of H2.

Structural and Biochemical Characterization of Spa47 Provides Mechanistic Insight into Type III Secretion System ATPase Activation and Shigella Virulence Regulation.

Like many Gram-negative pathogens, Shigella rely on a complex type III secretion system (T3SS) to inject effector proteins into host cells, take over host functions, and ultimately establish infection. Despite these critical roles, the energetics and regulatory mechanisms controlling the T3SS and pathogen virulence remain largely unclear. In this study, we present a series of high resolution crystal structures of Spa47 and use the structures to model an activated Spa47 oligomer, finding that ATP hydrolysis may be supported by specific side chain contributions from adjacent protomers within the complex. Follow-up mutagenesis experiments targeting the predicted active site residues validate the oligomeric model and determined that each of the tested residues are essential for Spa47 ATPase activity, although they are not directly responsible for stable oligomer formation. Although N-terminal domain truncation was necessary for crystal formation, it resulted in strictly monomeric Spa47 that is unable to hydrolyze ATP, despite maintaining the canonical ATPase core structure and active site residues. Coupled with studies of ATPase inactive full-length Spa47 point mutants, we find that Spa47 oligomerization and ATP hydrolysis are needed for complete T3SS apparatus formation, a proper translocator secretion profile, and Shigella virulence. This work represents the first structure-function characterization of Spa47, uniquely complementing the multitude of included Shigella T3SS phenotype assays and providing a more complete understanding of T3SS ATPase-mediated pathogen virulence. Additionally, these findings provide a strong platform for follow-up studies evaluating regulation of Spa47 oligomerization in vivo as a much needed means of treating and perhaps preventing shigellosis.