VNP20009 is a very effective anti-cancer agent and can specifically target tumors and inhibit tumor growth. It was assumed that the tumor targeting ability of VNP20009 correlated to its anticancer capacity. However, our observation contradicted to this assumption. Three VNP20009 mutant strains (ΔslyA, ΔSTM3120 and ΔhtrA) with reduced fitness in normal tissues and unchanged fitness in tumors partially or completely lost their anti-cancer capacities. The genes slyA, STM3120 and htrA were required for survival within macrophages and were indispensable for tumor microenvironment remodeling by VNP20009. The infiltration of immune cells occurred less in the tumors of mice infected with the mutant strains. In addition, the mRNA levels of TNF-α and IL-1β were significantly decreased in the tumors of mice treated with the mutant strains. Our results indicate that the immune responses elicited by bacteria rather than the bacterial titer in tumors play a “decisive” role in VNP20009-mediated bacterial cancer therapy, which provides a novel perspective for the underlying mechanism of bacterial cancer therapy.

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.

Megacities are major sources of anthropogenic fossil fuel CO₂(FFCO₂) emissions. The spatial extents of these large urban systems cover areas of 10 000 km² or more with complex topography and changing landscapes. We present a high-resolution land–atmosphere modelling system for urban CO₂ emissions over the Los Angeles (LA) megacity area. The Weather Research and Forecasting (WRF)-Chem model was coupled to a very high-resolution FFCO₂ emission product, Hestia-LA, to simulate atmospheric CO₂ concentrations across the LA megacity at spatial resolutions as fine as  ∼  1 km. We evaluated multiple WRF configurations, selecting one that minimized errors in wind speed, wind direction, and boundary layer height as evaluated by its performance against meteorological data collected during the CalNex-LA campaign (May–June 2010). Our results show no significant difference between moderate-resolution (4 km) and high-resolution (1.3 km) simulations when evaluated against surface meteorological data, but the high-resolution configurations better resolved planetary boundary layer heights and vertical gradients in the horizontal mean winds. We coupled our WRF configuration with the Vulcan 2.2 (10 km resolution) and Hestia-LA (1.3 km resolution) fossil fuel CO₂ emission products to evaluate the impact of the spatial resolution of the CO₂ emission products and the meteorological transport model on the representation of spatiotemporal variability in simulated atmospheric CO₂ concentrations. We find that high spatial resolution in the fossil fuel CO₂ emissions is more important than in the atmospheric model to capture CO₂ concentration variability across the LA megacity. Finally, we present a novel approach that employs simultaneous correlations of the simulated atmospheric CO₂ fields to qualitatively evaluate the greenhouse gas measurement network over the LA megacity. Spatial correlations in the atmospheric CO₂ fields reflect the coverage of individual measurement sites when a statistically significant number of sites observe emissions from a specific source or location. We conclude that elevated atmospheric CO₂ concentrations over the LA megacity are composed of multiple fine-scale plumes rather than a single homogenous urban dome. Furthermore, we conclude that FFCO₂ emissions monitoring in the LA megacity requires FFCO₂ emissions modelling with  ∼1 km resolution because coarser-resolution emissions modelling tends to overestimate the observational constraints on the emissions estimates.

Photoautotrophs assimilate oxidized carbon obtained from one of two sources: dissolved or atmospheric. Despite its size, the pool of lithospheric carbonate is not known to be a direct source for autotrophy. Yet, the mechanism that euendolithic cyanobacteria use to excavate solid carbonates suggests that minerals could directly supply CO[subscript 2] for autotrophy. Here, we use stable isotopes and NanoSIMS to show that the cyanobacterium Mastigocoleus testarum derives most of its carbon from the mineral it excavates, growing preferentially as an endolith when lacking dissolved CO[subscript 2]. Furthermore, natural endolithic communities from intertidal marine carbonate outcrops present carbon isotopic signatures consistent with mineral-sourced autotrophy. These data demonstrate a direct geomicrobial link between mineral carbonate pools and reduced organic carbon, which, given the geographical extent of carbonate outcrops, is likely of global relevance. The ancient fossil record of euendolithic cyanobacteria suggests that biological fixation of solid carbonate could have been relevant since the mid-Proterozoic.

We present a high-resolution atmospheric inversion system combining a Lagrangian Particle Dispersion Model (LPDM) and the Weather Research and Forecasting model (WRF), and test the impact of assimilating meteorological observation on transport accuracy. A Four Dimensional Data Assimilation (FDDA) technique continuously assimilates meteorological observations from various observing systems into the transport modeling system, and is coupled to the high resolution CO₂ emission product Hestia to simulate the atmospheric mole fractions of CO₂. For the Indianapolis Flux Experiment (INFLUX) project, we evaluated the impact of assimilating different meteorological observation systems on the linearized adjoint solutions and the CO₂ inverse fluxes estimated using observed CO₂ mole fractions from 11 out of 12 communications towers over Indianapolis for the Sep.-Nov. 2013 period. While assimilating WMO surface measurements improved the simulated wind speed and direction, their impact on the planetary boundary layer (PBL) was limited. Simulated PBL wind statistics improved significantly when assimilating upper-air observations from the commercial airline program Aircraft Communications Addressing and Reporting System (ACARS) and continuous ground-based Doppler lidar wind observations. Wind direction mean absolute error (MAE) decreased from 26 to 14 degrees and the wind speed MAE decreased from 2.0 to 1.2 m s_-1, while the bias remains small in all configurations (< 6 degrees and 0.2 m s_-1). Wind speed MAE and ME are larger in daytime than in nighttime. PBL depth MAE is reduced by ~10%, with little bias reduction. The inverse results indicate that the spatial distribution of CO₂ inverse fluxes were affected by the model performance while the overall flux estimates changed little across WRF simulations when aggregated over the entire domain. Our results show that PBL wind observations are a potent tool for increasing the precision of urban meteorological reanalyses, but that the impact on inverse flux estimates is dependent on the specific urban environment.

Butyrylcholinesterase (BChE) is an enzyme with broad substrate and ligand specificities and may function as a generalized bioscavenger by binding and/or hydrolyzing various xenobiotic agents and toxicants, many of which target the central and peripheral nervous systems. Variants of BChE were rationally designed to increase the enzyme’s ability to hydrolyze the psychoactive enantiomer of cocaine. These variants were cloned, and then expressed using the magnICON transient expression system in plants and their enzymatic properties were investigated. In particular, we explored the effects that these site-directed mutations have over the enzyme kinetics with various substrates of BChE. We further compared the affinity of various anticholinesterases including organophosphorous nerve agents and pesticides toward these BChE variants relative to the wild type enzyme. In addition to serving as a therapy for cocaine addiction-related diseases, enhanced bioscavenging against other harmful agents could add to the practicality and versatility of the plant-derived recombinant enzyme as a multivalent therapeutic.

The INFLUX experiment has taken multiple approaches to estimate the carbon dioxide (CO₂) flux in a domain centered on the city of Indianapolis, Indiana. One approach, Hestia, uses a bottom-up technique relying on a mixture of activity data, fuel statistics, direct flux measurement and modeling algorithms. A second uses a Bayesian atmospheric inverse approach constrained by atmospheric CO₂ measurements and the Hestia emissions estimate as a prior CO₂ flux. The difference in the central estimate of the two approaches comes to 0.94 MtC (an 18.7% difference) over the eight-month period between September 1, 2012 and April 30, 2013, a statistically significant difference at the 2-sigma level. Here we explore possible explanations for this apparent discrepancy in an attempt to reconcile the flux estimates. We focus on two broad categories: 1) biases in the largest of bottom-up flux contributions and 2) missing CO₂ sources. Though there is some evidence for small biases in the Hestia fossil fuel carbon dioxide (FFCO2) flux estimate as an explanation for the calculated difference, we find more support for missing CO₂ fluxes, with biological respiration the largest of these. Incorporation of these differences bring the Hestia bottom-up and the INFLUX inversion flux estimates into statistical agreement and are additionally consistent with wintertime measurements of atmospheric ¹⁴CO₂. We conclude that comparison of bottom-up and top-down approaches must consider all flux contributions and highlight the important contribution to urban carbon budgets of animal and biotic respiration. Incorporation of missing CO₂ fluxes reconciles the bottom-up and inverse-based approach in the INFLUX domain.