High-throughput quantification of microbial birth and death dynamics using fluorescence microscopy

Background: Microbes live in dynamic environments where nutrient concentrations fluctuate. Quantifying fitness in terms of birth rate and death rate in a wide range of environments is critical for understanding microbial evolution and ecology. Methods: Here, using high-throughput time-lapse microscopy, we have quantified how Saccharomyces cerevisiae mutants incapable of synthesizing an essential metabolite (auxotrophs) grow or die in various concentrations of the required metabolite. We establish that cells normally expressing fluorescent proteins lose fluorescence upon death and that the total fluorescence in an imaging frame is proportional to the number of live cells even when cells form multiple layers. We validate our microscopy approach of measuring birth and death rates using flow cytometry, cell counting, and chemostat culturing. Results: For lysine-requiring cells, very low concentrations of lysine are not detectably consumed and do not support cell birth, but delay the onset of death phase and reduce the death rate compared to no lysine. In contrast, in low hypoxanthine, hypoxanthine-requiring cells can produce new cells, yet also die faster than in the absence of hypoxanthine. For both strains, birth rates under various metabolite concentrations are better described by the sigmoidal-shaped Moser model than the well-known Monod model, while death rates can vary with metabolite concentration and time. Conclusions: Our work reveals how time-lapse microscopy can be used to discover non-intuitive microbial birth and death dynamics and to quantify growth rates in many environments.     

Accurate,Fully-Automated NMR Spectral Profiling for Metabolomics

Many diseases cause significant changes to the concentrations of small molecules (a.k.a. metabolites) that appear in a person’s biofluids, which means such diseases can often be readily detected from a person’s “metabolic profile"—i.e., the list of concentrations of those metabolites. This information can be extracted from a biofluids Nuclear Magnetic Resonance (NMR) spectrum. However, due to its complexity, NMR spectral profiling has remained manual, resulting in slow, expensive and error-prone procedures that have hindered clinical and industrial adoption of metabolomics via NMR. This paper presents a system, BAYESIL, which can quickly, accurately, and autonomously produce a person’s metabolic profile. Given a 1D ¹H NMR spectrum of a complex biofluid (specifically serum or cerebrospinal fluid), BAYESIL can automatically determine the metabolic profile. This requires first performing several spectral processing steps, then matching the resulting spectrum against a reference compound library, which contains the “signatures” of each relevant metabolite. BAYESIL views spectral matching as an inference problem within a probabilistic graphical model that rapidly approximates the most probable metabolic profile. Our extensive studies on a diverse set of complex mixtures including real biological samples (serum and CSF), defined mixtures and realistic computer generated spectra; involving > 50 compounds, show that BAYESIL can autonomously find the concentration of NMR-detectable metabolites accurately (~ 90% correct identification and ~ 10% quantification error), in less than 5 minutes on a single CPU. These results demonstrate that BAYESIL is the first fully-automatic publicly-accessible system that provides quantitative NMR spectral profiling effectively—with an accuracy on these biofluids that meets or exceeds the performance of trained experts. We anticipate this tool will usher in high-throughput metabolomics and enable a wealth of new applications of NMR in clinical settings. BAYESIL is accessible at http://www.bayesil.ca.     

Mechanisms of Human Erythrocytic Bioactivation of Nitrite

Nitrite signaling likely occurs through its reduction to nitric oxide (NO). Several reports support a role of erythrocytes and hemoglobin in nitrite reduction, but this remains controversial, and alternative reductive pathways have been proposed. In this work we determined whether the primary human erythrocytic nitrite reductase is hemoglobin as opposed to other erythrocytic proteins that have been suggested to be the major source of nitrite reduction. We employed several different assays to determine NO production from nitrite in erythrocytes including electron paramagnetic resonance detection of nitrosyl hemoglobin, chemiluminescent detection of NO, and inhibition of platelet activation and aggregation. Our studies show that NO is formed by red blood cells and inhibits platelet activation. Nitric oxide formation and signaling can be recapitulated with isolated deoxyhemoglobin. Importantly, there is limited NO production from erythrocytic xanthine oxidoreductase and nitric-oxide synthase. Under certain conditions we find dorzolamide (an inhibitor of carbonic anhydrase) results in diminished nitrite bioactivation, but the role of carbonic anhydrase is abrogated when physiological concentrations of CO₂ are present. Importantly, carbon monoxide, which inhibits hemoglobin function as a nitrite reductase, abolishes nitrite bioactivation. Overall our data suggest that deoxyhemoglobin is the primary erythrocytic nitrite reductase operating under physiological conditions and accounts for nitrite-mediated NO signaling in blood.     

The EF-Hand Ca2+ Binding Protein MICU Choreographs Mitochondrial Ca2+ Dynamics in Arabidopsis

Plant organelle function must constantly adjust to environmental conditions, which requires dynamic coordination. Ca²⁺ signaling may play a central role in this process. Free Ca²⁺ dynamics are tightly regulated and differ markedly between the cytosol, plastid stroma, and mitochondrial matrix. The mechanistic basis of compartment-specific Ca²⁺ dynamics is poorly understood. Here, we studied the function of At-MICU, an EF-hand protein of Arabidopsis thaliana with homology to constituents of the mitochondrial Ca²⁺ uniporter machinery in mammals. MICU binds Ca²⁺ and localizes to the mitochondria in Arabidopsis. In vivo imaging of roots expressing a genetically encoded Ca²⁺ sensor in the mitochondrial matrix revealed that lack of MICU increased resting concentrations of free Ca²⁺ in the matrix. Furthermore, Ca²⁺ elevations triggered by auxin and extracellular ATP occurred more rapidly and reached higher maximal concentrations in the mitochondria of micu mutants, whereas cytosolic Ca²⁺ signatures remained unchanged. These findings support the idea that a conserved uniporter system, with composition and regulation distinct from the mammalian machinery, mediates mitochondrial Ca²⁺ uptake in plants under in vivo conditions. They further suggest that MICU acts as a throttle that controls Ca²⁺ uptake by moderating influx, thereby shaping Ca²⁺ signatures in the matrix and preserving mitochondrial homeostasis. Our results open the door to genetic dissection of mitochondrial Ca²⁺ signaling in plants.     

Efficacy of Fluensulfone in a Tomato–Cucumber Double Cropping System

Vegetable crops in the southeastern United States are commonly grown on plastic mulch with two crop cycles produced on a single mulch application. Field trials were conducted in 2013 and 2014 in two locations to evaluate the efficacy of fluensulfone for controlling Meloidogyne spp. when applied through drip irrigation to cucumber in a tomato–cucumber double-cropping system. In the spring tomato crop, 1,3-dichloropropene (1,3-D), fluensulfone, and a resistant cultivar significantly decreased root galling by 91%, 73%, and 97%, respectively, compared to the untreated control. Tomato plots from the spring were divided into split plots for the fall where the main plots were the spring treatment and the subplots were cucumber either treated with fluensulfone (3.0 kg a.i./ha. via drip irrigation) or left untreated. The fall application of fluensulfone improved cucumber vigor and reduced gall ratings compared to untreated subplots. Fluensulfone reduced damage from root-knot nematodes when applied to the first crop as well as provided additional protection to the second crop when it was applied through a drip system.     

Wind‐mediated horseweed (Conyza canadensis) gene flow: pollen emission,dispersion, and deposition

Horseweed (Conyza canadensis) is a problem weed in crop production because of its evolved resistance to glyphosate and other herbicides. Although horseweed is mainly self-pollinating, glyphosate-resistant (GR) horseweed can pollinate glyphosate-susceptible (GS) horseweed. To the best of our knowledge, however, there are no available data on horseweed pollen production, dispersion, and deposition relative to gene flow and the evolution of resistance. To help fill this knowledge gap, a 43-day field study was performed in Champaign, Illinois, USA in 2013 to characterize horseweed atmospheric pollen emission, dispersion, and deposition. Pollen concentration and deposition, coupled with atmospheric data, were measured in a source field (180 m by 46 m) and its surrounding areas up to 1 km downwind horizontally and up to 100 m vertically. The source strength (emission rate) ranged from 0 to 140 pollen grains per plant per second (1170 to 2.1×10⁶ per plant per day). For the life of the study, the estimated number of pollen grains generated from this source field was 10.5×10¹⁰ (2.3×10⁶ per plant). The release of horseweed pollen was not strongly correlated to meteorological data and may be mainly determined by horseweed physiology. Horseweed pollen reached heights of 80 to100 m, making long-distance transport possible. Normalized (by source data) pollen deposition with distance followed a negative-power exponential curve. Normalized pollen deposition was 2.5% even at 480 m downwind from the source edge. Correlation analysis showed that close to or inside the source field at lower heights (≤3 m) vertical transport was related to vertical wind speed, while horizontal pollen transport was related to horizontal wind speed. High relative humidity prevented pollen transport at greater heights (3–100 m) and longer distances (0–1000 m) from the source. This study can contribute to the understanding of how herbicide-resistance weeds or invasive plants affect ecology through wind-mediated pollination and invasion.     

Ultraviolet Radiation Influences Perch Selection by a Neotropical Poison-Dart Frog

Ambient ultraviolet-B radiation can harm amphibian eggs, larvae and adults. However, some amphibians avoid UV-B radiation when given the opportunity. The strawberry poison dart frog, Oophaga pumilio, is diurnal and males vocalize throughout the day in light gaps under forest canopies that expose them to solar radiation. Previous studies have demonstrated that males calling from high perches are more successful at mating than those at lower perches. We investigated whether frogs at higher perches receive more ultraviolet-B than those calling from lower perches. We also investigated whether frogs on perches receiving relatively low ultraviolet-B levels maintained their positions for longer compared to individuals calling from perches receiving higher levels of ultraviolet-B. Finally, since it has been hypothesized that some animals utilize levels of UV-A as a visual cue to avoid UV-B damage, we artificially elevated ultraviolet-A levels to examine whether males exposed to artificially elevated ultraviolet-A abandoned their perches sooner compared to males exposed to visible light. We found that frogs called from perches receiving low ultraviolet-B regardless of perch height, and that frogs maintain their positions longer on perches receiving low ultraviolet-B compared to perches receiving even slightly higher ultraviolet-B levels. Exposing the frogs to artificially elevated levels of ultraviolet-A radiation caused males to move off of their perches faster than when they were exposed to a control light source. These experiments suggest that ultraviolet radiation plays an important role in frog behavior related to perch selection, even in rainforests where much of the solar radiation is shielded by the forest canopy.     

The macroecology of infectious diseases: a new perspective on global‐scale drivers of pathogen distributions and impacts

Identifying drivers of infectious disease patterns and impacts at the broadest scales of organisation is one of the most crucial challenges for modern science, yet answers to many fundamental questions remain elusive. These include what factors commonly facilitate transmission of pathogens to novel host species, what drives variation in immune investment among host species, and more generally what drives global patterns of parasite diversity and distribution? Here we consider how the perspectives and tools of macroecology, a field that investigates patterns and processes at broad spatial, temporal and taxonomic scales, are expanding scientific understanding of global infectious disease ecology. In particular, emerging approaches are providing new insights about scaling properties across all living taxa, and new strategies for mapping pathogen biodiversity and infection risk. Ultimately, macroecology is establishing a framework to more accurately predict global patterns of infectious disease distribution and emergence.