ASU Scholarship Showcase

Photosynthesis, a process catalysed by plants, algae and cyanobacteria converts sunlight to energy thus sustaining all higher life on Earth. Two large membrane protein complexes, photosystem I and II (PSI and PSII), act in series to catalyse the light-driven reactions in photosynthesis. PSII catalyses the light-driven water splitting process, which maintains the Earth’s oxygenic atmosphere. In this process, the oxygen-evolving complex (OEC) of PSII cycles through five states, S₀ to S₄, in which four electrons are sequentially extracted from the OEC in four light-driven charge-separation events. Here we describe time resolved experiments on PSII nano/microcrystals from Thermosynechococcus elongatus performed with the recently developed technique of serial femtosecond crystallography. Structures have been determined from PSII in the dark S₁ state and after double laser excitation (putative S₃ state) at 5 and 5.5 Å resolution, respectively. The results provide evidence that PSII undergoes significant conformational changes at the electron acceptor side and at the Mn₄CaO₅ core of the OEC. These include an elongation of the metal cluster, accompanied by changes in the protein environment, which could allow for binding of the second substrate water molecule between the more distant protruding Mn (referred to as the ‘dangler’ Mn) and the Mn₃CaO_x cubane in the S₂ to S₃ transition, as predicted by spectroscopic and computational studies. This work shows the great potential for time-resolved serial femtosecond crystallography for investigation of catalytic processes in biomolecules.

We present results from experiments at the Linac Coherent Light Source (LCLS) demonstrating that serial femtosecond crystallography (SFX) can be performed to high resolution (~2.5 Å) using protein microcrystals deposited on an ultra-thin silicon nitride membrane and embedded in a preservation medium at room temperature. Data can be acquired at a high acquisition rate using x-ray free electron laser sources to overcome radiation damage, while sample consumption is dramatically reduced compared to flowing jet methods. We achieved a peak data acquisition rate of 10 Hz with a hit rate of ~38%, indicating that a complete data set could be acquired in about one 12-hour LCLS shift using the setup described here, or in even less time using hardware optimized for fixed target SFX. This demonstration opens the door to ultra low sample consumption SFX using the technique of diffraction-before-destruction on proteins that exist in only small quantities and/or do not produce the copious quantities of microcrystals required for flowing jet methods.

Previous proof-of-concept measurements on single-layer two-dimensional membrane-protein crystals performed at X-ray free-electron lasers (FELs) have demonstrated that the collection of meaningful diffraction patterns, which is not possible at synchrotrons because of radiation-damage issues, is feasible. Here, the results obtained from the analysis of a thousand single-shot, room-temperature X-ray FEL diffraction images from two-dimensional crystals of a bacteriorhodopsin mutant are reported in detail. The high redundancy in the measurements boosts the intensity signal-to-noise ratio, so that the values of the diffracted intensities can be reliably determined down to the detector-edge resolution of 4 Å. The results show that two-dimensional serial crystallography at X-ray FELs is a suitable method to study membrane proteins to near-atomic length scales at ambient temperature. The method presented here can be extended to pump–probe studies of optically triggered structural changes on submillisecond timescales in two-dimensional crystals, which allow functionally relevant large-scale motions that may be quenched in three-dimensional crystals.

The probiotic effects of Lactobacillus reuteri have been speculated to partly depend on its capacity to produce the antimicrobial substance reuterin during the reduction of glycerol in the gut. In this study, the potential of this process to protect human intestinal epithelial cells against infection with Salmonella enterica serovar Typhimurium was investigated. We used a three-dimensional (3-D) organotypic model of human colonic epithelium that was previously validated and applied to study interactions between S. Typhimurium and the intestinal epithelium that lead to enteric salmonellosis. Using this model system, we show that L. reuteri protects the intestinal cells against the early stages of Salmonella infection and that this effect is significantly increased when L. reuteri is stimulated to produce reuterin from glycerol. More specifically, the reuterin-containing ferment of L. reuteri caused a reduction in Salmonella adherence and invasion (1 log unit), and intracellular survival (2 log units). In contrast, the L. reuteri ferment without reuterin stimulated growth of the intracellular Salmonella population with 1 log unit. The short-term exposure to reuterin or the reuterin-containing ferment had no observed negative impact on intestinal epithelial cell health. However, long-term exposure (24 h) induced a complete loss of cell-cell contact within the epithelial aggregates and compromised cell viability. Collectively, these results shed light on a potential role for reuterin in inhibiting Salmonella-induced intestinal infections and may support the combined application of glycerol and L. reuteri. While future in vitro and in vivo studies of reuterin on intestinal health should fine-tune our understanding of the mechanistic effects, in particular in the presence of a complex gut microbiota, this the first report of a reuterin effect on the enteric infection process in any mammalian cell type.

Extra-intestinal pathogenic E. coli (ExPEC), including avian pathogenic E. coli (APEC), pose a considerable threat to both human and animal health, with illness causing substantial economic loss. APEC strain χ7122 (O78∶K80∶H9), containing three large plasmids [pChi7122-1 (IncFIB/FIIA-FIC), pChi7122-2 (IncFII), and pChi7122-3 (IncI₂)]; and a small plasmid pChi7122-4 (ColE2-like), has been used for many years as a model strain to study the molecular mechanisms of ExPEC pathogenicity and zoonotic potential. We previously sequenced and characterized the plasmid pChi7122-1 and determined its importance in systemic APEC infection; however the roles of the other pChi7122 plasmids were still ambiguous. Herein we present the sequence of the remaining pChi7122 plasmids, confirming that pChi7122-2 and pChi7122-3 encode an ABC iron transport system (eitABCD) and a putative type IV fimbriae respectively, whereas pChi7122-4 is a cryptic plasmid. New features were also identified, including a gene cluster on pChi7122-2 that is not present in other E. coli strains but is found in Salmonella serovars and is predicted to encode the sugars catabolic pathways. In vitro evaluation of the APEC χ7122 derivative strains with the three large plasmids, either individually or in combinations, provided new insights into the role of plasmids in biofilm formation, bile and acid tolerance, and the interaction of E. coli strains with 3-D cultures of intestinal epithelial cells. In this study, we show that the nature and combinations of plasmids, as well as the background of the host strains, have an effect on these phenomena. Our data reveal new insights into the role of extra-chromosomal sequences in fitness and diversity of ExPEC in their phenotypes.

Strategies are needed to improve repopulation of decellularized lung scaffolds with stromal and functional epithelial cells. We demonstrate that decellularized mouse lungs recellularized in a dynamic low fluid shear suspension bioreactor, termed the rotating wall vessel (RWV), contained more cells with decreased apoptosis, increased proliferation and enhanced levels of total RNA compared to static recellularization conditions. These results were observed with two relevant mouse cell types: bone marrow-derived mesenchymal stromal (stem) cells (MSCs) and alveolar type II cells (C10). In addition, MSCs cultured in decellularized lungs under static but not bioreactor conditions formed multilayered aggregates. Gene expression and immunohistochemical analyses suggested differentiation of MSCs into collagen I-producing fibroblast-like cells in the bioreactor, indicating enhanced potential for remodeling of the decellularized scaffold matrix. In conclusion, dynamic suspension culture is promising for enhancing repopulation of decellularized lungs, and could contribute to remodeling the extracellular matrix of the scaffolds with subsequent effects on differentiation and functionality of inoculated cells.

Land surface energy balance in a built environment is widely modelled using urban canopy models with representation of building arrays as big street canyons. Modification of this simplified geometric representation, however, leads to challenging numerical difficulties in improving physical parameterization schemes that are deterministic in nature. In this paper, we develop a stochastic algorithm to estimate view factors between canyon facets in the presence of shade trees based on Monte Carlo simulation, where an analytical formulation is inhibited by the complex geometry. The model is validated against analytical solutions of benchmark radiative problems as well as field measurements in real street canyons. In conjunction with the matrix method resolving infinite number of reflections, the proposed model is capable of predicting the radiative exchange inside the street canyon with good accuracy. Modeling of transient evolution of thermal filed inside the street canyon using the proposed method demonstrate the potential of shade trees in mitigating canyon surface temperatures as well as saving of building energy use. This new numerical framework also deepens our insight into the fundamental physics of radiative heat transfer and surface energy balance for urban climate modeling.

Studies on urban heat island (UHI) have been more than a century after the phenomenon was first discovered in the early 1800s. UHI emerges as the source of many urban environmental problems and exacerbates the living environment in cities. Under the challenges of increasing urbanization and future climate changes, there is a pressing need for sustainable adaptation/mitigation strategies for UHI effects, one popular option being the use of reflective materials. While it is introduced as an effective method to reduce temperature and energy consumption in cities, its impacts on environmental sustainability and large-scale non-local effect are inadequately explored. This paper provides a synthetic overview of potential environmental impacts of reflective materials at a variety of scales, ranging from energy load on a single building to regional hydroclimate. The review shows that mitigation potential of reflective materials depends on a set of factors, including building characteristics, urban environment, meteorological and geographical conditions, to name a few. Precaution needs to be exercised by city planners and policy makers for large-scale deployment of reflective materials before their environmental impacts, especially on regional hydroclimates, are better understood. In general, it is recommended that optimal strategy for UHI needs to be determined on a city-by-city basis, rather than adopting a “one-solution-fits-all” strategy.

It has been suggested that the extended intensity profiles surrounding Bragg reflections that arise when a series of finite crystals of varying size and shape are illuminated by the intense, coherent illumination of an x-ray free-electron laser may enable the crystal’s unit-cell electron density to be obtained ab initio via well-established iterative phasing algorithms. Such a technique could have a significant impact on the field of biological structure determination since it avoids the need for a priori information from similar known structures, multiple measurements near resonant atomic absorption energies, isomorphic derivative crystals, or atomic-resolution data. Here, we demonstrate this phasing technique on diffraction patterns recorded from artificial two-dimensional microcrystals using the seeded soft x-ray free-electron laser FERMI. We show that the technique is effective when the illuminating wavefront has nonuniform phase and amplitude, and when the diffraction intensities cannot be measured uniformly throughout reciprocal space because of a limited signal-to-noise ratio.

The hysteresis effect in diurnal cycles of net radiation R-n and ground heat flux G(0) has been observed in many studies, while the governing mechanism remains vague. In this study, we link the phenomenology of hysteresis loops to the wave phase difference between the diurnal evolutions of various terms in the surface energy balance. R-n and G(0) are parameterized with the incoming solar radiation and the surface temperature as two control parameters of the surface energy partitioning. The theoretical analysis shows that the vertical water flux W and the scaled ratio A(s)*/A(T)* (net shortwave radiation to outgoing longwave radiation) play crucial roles in shaping hysteresis loops of R-n and G(0). Comparisons to field measurements indicate that hysteresis loops for different land covers can be well captured by the theoretical model, which is also consistent with Camuffo-Bernadi formula. This study provides insight into the surface partitioning and temporal evolution of the energy budget at the land surface.