Three-dimensional models of human intestinal epithelium mimic the differentiated form and function of parental tissues often not exhibited by two-dimensional monolayers and respond to Salmonella in key ways that reflect in vivo infections. To further enhance the physiological relevance of three-dimensional models to more closely approximate in vivo intestinal microenvironments encountered by Salmonella, we developed and validated a novel three-dimensional co-culture infection model of colonic epithelial cells and macrophages using the NASA Rotating Wall Vessel bioreactor. First, U937 cells were activated upon collagen-coated scaffolds. HT-29 epithelial cells were then added and the three-dimensional model was cultured in the bioreactor until optimal differentiation was reached, as assessed by immunohistochemical profiling and bead uptake assays. The new co-culture model exhibited in vivo-like structural and phenotypic characteristics, including three-dimensional architecture, apical-basolateral polarity, well-formed tight/adherens junctions, mucin, multiple epithelial cell types, and functional macrophages. Phagocytic activity of macrophages was confirmed by uptake of inert, bacteria-sized beads. Contribution of macrophages to infection was assessed by colonization studies of Salmonella pathovars with different host adaptations and disease phenotypes (Typhimurium ST19 strain SL1344 and ST313 strain D23580; Typhi Ty2). In addition, Salmonella were cultured aerobically or microaerobically, recapitulating environments encountered prior to and during intestinal infection, respectively. All Salmonella strains exhibited decreased colonization in co-culture (HT-29-U937) relative to epithelial (HT-29) models, indicating antimicrobial function of macrophages. Interestingly, D23580 exhibited enhanced replication/survival in both models following invasion. Pathovar-specific differences in colonization and intracellular co-localization patterns were observed. These findings emphasize the power of incorporating a series of related three-dimensional models within a study to identify microenvironmental factors important for regulating infection.

Nonhyperbolicity, as characterized by the coexistence of Kolmogorov-Arnold-Moser (KAM) tori and chaos in the phase space, is generic in classical Hamiltonian systems. An open but fundamental question in physics concerns the relativistic quantum manifestations of nonhyperbolic dynamics. We choose the mushroom billiard that has been mathematically proven to be nonhyperbolic, and study the resonant tunneling dynamics of a massless Dirac fermion. We find that the tunneling rate as a function of the energy exhibits a striking "clustering" phenomenon, where the majority of the values of the rate concentrate on a narrow region, as a result of the chaos component in the classical phase space. Relatively few values of the tunneling rate, however, spread outside the clustering region due to the integrable component. Resonant tunneling of electrons in nonhyperbolic chaotic graphene systems exhibits a similar behavior. To understand these numerical results, we develop a theoretical framework by combining analytic solutions of the Dirac equation in certain integrable domains and physical intuitions gained from current understanding of the quantum manifestations of chaos. In particular, we employ a theoretical formalism based on the concept of self-energies to calculate the tunneling rate and analytically solve the Dirac equation in one dimension as well as in two dimensions for a circular-ring-type of tunneling systems exhibiting integrable dynamics in the classical limit. Because relatively few and distinct classical periodic orbits are present in the integrable component, the corresponding relativistic quantum states can have drastically different behaviors, leading to a wide spread in the values of the tunneling rate in the energy-rate plane. In contrast, the chaotic component has embedded within itself an infinite number of unstable periodic orbits, which provide far more quantum states for tunneling. Due to the nature of chaos, these states are characteristically similar, leading to clustering of the values of the tunneling rate in a narrow band. The appealing characteristic of our work is a demonstration and physical understanding of the "mixed" role played by chaos and regular dynamics in shaping relativistic quantum tunneling dynamics.

Antibiotic-resistant bacterial infections are difficult to treat, producing a burden on healthcare and the economy. Extraintestinal pathogenic Escherichia coli (ExPEC) strains frequently carry antibiotic resistance genes, cause infections outside of the intestine, and are causative agents of hospital-acquired infections. Developing a prevention strategy against this pathogen is challenging due to its antibiotic resistance and antigenic diversity. E. coli common pilus (ECP) is frequently found in ExPEC strains and may serve as a common antigen to induce protection against several ExPEC serotypes. In addition, live recombinant attenuated Salmonella vaccine (RASV) strains have been used to prevent Salmonella infection and can also be modified to deliver foreign antigens. Thus, the objective of this study was to design a RASV to produce ECP on its surface and assess its ability to provide protection against ExPEC infections. To constitutively display ECP in a RASV strain, we genetically engineered a vector (pYA4428) containing aspartate-β-semialdehyde dehydrogenase and E. coli ecp genes and introduced it into RASV χ9558. RASV χ9558 containing an empty vector (pYA3337) was used as a control to assess protection conferred by the RASV strain without ECP.

We assessed vaccine efficacy in in vitro bacterial inhibition assays and mouse models of ExPEC-associated human infections. We found that RASV χ9558(pYA4428) synthesized the major pilin (EcpA) and tip pilus adhesin (EcpD) on the bacterial surface. Mice orally vaccinated with RASV χ9558(pYA3337) without ECP or χ9558(pYA4428) with ECP, produced anti-Salmonella LPS and anti-E. coli EcpA and EcpD IgG and IgA antibodies. RASV strains showed protective potential against some E. coli and Salmonella strains as assessed using in vitro assays. In mouse sepsis and urinary tract infection challenge models, both vaccines had significant protection in some internal organs. Overall, this work showed that RASVs can elicit an immune response to E. coli and Salmonella antigens in some mice, provide significant protection in some internal organs during ExPEC challenge, and thus this study is a promising initial step toward developing a vaccine for prevention of ExPEC infections. Future studies should optimize the ExPEC antigens displayed by the RASV strain for a more robust immune response and enhanced protection against ExPEC infection.

A tetradentate Pd(II) complex, Pd3O3, which exhibits highly efficient excimer emission is synthesized and characterized. Pd3O3 can achieve blue emission despite using phenyl-pyridine emissive ligands which have been a mainstay of stable green and red phosphorescent emitter designs, making Pd3O3 a good candidate for stable blue or white OLEDs. Pd3O3 exhibits strong and efficient phosphorescent excimer emission expanding the excimer based white OLEDs beyond the sole class of Pt complexes. Devices of Pd3O3 demonstrate peak external quantum efficiencies as high as 24.2% and power efficiencies of 67.9 Lm per W for warm white devices. Furthermore, Pd3O3 devices in a carefully designed stable structure achieved a device operational lifetime of nearly 3000 h at 1000 cd m^-2 without any outcoupling enhancement while simultaneously achieving peak external quantum efficiencies of 27.3% and power efficiencies over 81 Lm per W.

Low fluid shear force, including that encountered in microgravity models, induces bacterial responses, but the range of bacteria capable of responding to this signal remains poorly characterized. We systematically analyzed a range of Gram negative Enterobacteriaceae for conservation of the low-shear modeled microgravity (LSMMG) response using phenotypic assays, qPCR, and targeted mutations. Our results indicate LSMMG response conservation across Enterobacteriacae with potential variance in up- or down-regulation of a given response depending on genus. Based on the data, we analyzed the role of the trp operon genes and the TrpR regulator in the LSMMG response using targeted mutations in these genes in S. Typhimurium and E. coli. We found no alteration of the LSMMG response compared to WT in these mutant strains under the conditions tested here. To our knowledge, this study is first-of-kind for Citrobacter, Enterobacter, and Serratia, presents novel data for Escherichia, and provides the first analysis of trp genes in LSMMG responses. This impacts our understanding of how LSMMG affects bacteria and our ability to modify bacteria with this condition in the future.

A distinct pathovar of Salmonella enterica serovar Typhimurium, ST313, has emerged in sub-Saharan Africa as a major cause of fatal bacteremia in young children and HIV-infected adults. D23580, a multidrug resistant clinical isolate of ST313, was previously shown to have undergone genome reduction in a manner that resembles that of the more human-restricted pathogen, Salmonella enterica serovar Typhi. It has since been shown through tissue distribution studies that D23580 is able to establish an invasive infection in chickens. However, it remains unclear whether ST313 can cause lethal disease in a non-human host following a natural course of infection. Herein we report that D23580 causes lethal and invasive disease in a murine model of infection following peroral challenge. The LD⁵⁰ of D23580 in female BALB/c mice was 4.7 x 10⁵ CFU. Tissue distribution studies performed 3 and 5 days post-infection confirmed that D23580 was able to more rapidly colonize the spleen, mesenteric lymph nodes and gall bladder in mice when compared to the well-characterized S. Typhimurium strain SL1344. D23580 exhibited enhanced resistance to acid stress relative to SL1344, which may lend towards increased capability to survive passage through the gastrointestinal tract as well as during its intracellular lifecycle. Interestingly, D23580 also displayed higher swimming motility relative to SL1344, S. Typhi strain Ty2, and the ST313 strain A130. Biochemical tests revealed that D23580 shares many similar metabolic features with SL1344, with several notable differences in the Voges-Proskauer and catalase tests, as well alterations in melibiose, and inositol utilization. These results represent the first full duration infection study using an ST313 strain following the entire natural course of disease progression, and serve as a benchmark for ongoing and future studies into the pathogenesis of D23580.

In vitro models that mimic in vivo host-pathogen interactions are needed to evaluate candidate drugs that inhibit bacterial virulence traits. We established a new approach to study Pseudomonas aeruginosa biofilm susceptibility on biotic surfaces, using a three-dimensional (3-D) lung epithelial cell model. P. aeruginosa formed antibiotic resistant biofilms on 3-D cells without affecting cell viability. The biofilm-inhibitory activity of antibiotics and/or the anti-biofilm peptide DJK-5 were evaluated on 3-D cells compared to a plastic surface, in medium with and without fetal bovine serum (FBS). In both media, aminoglycosides were more efficacious in the 3-D cell model. In serum-free medium, most antibiotics (except polymyxins) showed enhanced efficacy when 3-D cells were present. In medium with FBS, colistin was less efficacious in the 3-D cell model. DJK-5 exerted potent inhibition of P. aeruginosa association with both substrates, only in serum-free medium. DJK-5 showed stronger inhibitory activity against P. aeruginosa associated with plastic compared to 3-D cells. The combined addition of tobramycin and DJK-5 exhibited more potent ability to inhibit P. aeruginosa association with both substrates. In conclusion, lung epithelial cells influence the efficacy of most antimicrobials against P. aeruginosa biofilm formation, which in turn depends on the presence or absence of FBS.

Successful identification of directed dynamical influence in complex systems is relevant to significant problems of current interest. Traditional methods based on Granger causality and transfer entropy have issues such as difficulty with nonlinearity and large data requirement. Recently a framework based on nonlinear dynamical analysis was proposed to overcome these difficulties. We find, surprisingly, that noise can counterintuitively enhance the detectability of directed dynamical influence. In fact, intentionally injecting a proper amount of asymmetric noise into the available time series has the unexpected benefit of dramatically increasing confidence in ascertaining the directed dynamical influence in the underlying system. This result is established based on both real data and model time series from nonlinear ecosystems. We develop a physical understanding of the beneficial role of noise in enhancing detection of directed dynamical influence.

Despite serving as the primary entry portal for coxsackievirus B (CVB), little is known about CVB infection of the intestinal epithelium, owing at least in part to the lack of suitable in vivo models and the inability of cultured cells to recapitulate the complexity and structure associated with the gastrointestinal (GI) tract. Here, we report on the development of a three-dimensional (3-D) organotypic cell culture model of Caco-2 cells to model CVB infection of the gastrointestinal epithelium. We show that Caco-2 cells grown in 3-D using the rotating wall vessel (RWV) bioreactor recapitulate many of the properties of the intestinal epithelium, including the formation of well-developed tight junctions, apical-basolateral polarity, brush borders, and multicellular complexity. In addition, transcriptome analyses using transcriptome sequencing (RNA-Seq) revealed the induction of a number of genes associated with intestinal epithelial differentiation and/or intestinal processes in vivo when Caco-2 cells were cultured in 3-D. Applying this model to CVB infection, we found that although the levels of intracellular virus production were similar in two-dimensional (2-D) and 3-D Caco-2 cell cultures, the release of infectious CVB was enhanced in 3-D cultures at early stages of infection. Unlike CVB, the replication of poliovirus (PV) was significantly reduced in 3-D Caco-2 cell cultures. Collectively, our studies show that Caco-2 cells grown in 3-D using the RWV bioreactor provide a cell culture model that structurally and transcriptionally represents key aspects of cells in the human GI tract and can thus be used to expand our understanding of enterovirus-host interactions in intestinal epithelial cells.

In eutherians, the placenta acts as a barrier and conduit at the maternal-fetal interface. Syncytiotrophoblasts, the multinucleated cells that cover the placental villous tree surfaces of the human placenta, are directly bathed in maternal blood and are formed by the fusion of progenitor cytotrophoblasts that underlie them. Despite their crucial role in fetal protection, many of the events that govern trophoblast fusion and protection from microbial infection are unknown. We describe a three-dimensional (3D)–based culture model using human JEG-3 trophoblast cells that develop syncytiotrophoblast phenotypes when cocultured with human microvascular endothelial cells. JEG-3 cells cultured in this system exhibit enhanced fusogenic activity and morphological and secretory activities strikingly similar to those of primary human syncytiotrophoblasts. RNASeq analyses extend the observed functional similarities to the transcriptome, where we observed significant overlap between syncytiotrophoblast-specific genes and 3D JEG-3 cultures. Furthermore, JEG-3 cells cultured in 3D are resistant to infection by viruses and Toxoplasma gondii, which mimics the high resistance of syncytiotrophoblasts to microbial infections in vivo. Given that this system is genetically manipulatable, it provides a new platform to dissect the mechanisms involved in syncytiotrophoblast development and microbial resistance.