Extracting Transition Rates in Particle Tracking Using Analytical Diffusion Distribution Analysis

Single-particle tracking is an important technique in the life sciences to understand the kinetics of biomolecules. The analysis of apparent diffusion coefficients in vivo, for example, enables researchers to determine whether biomolecules are moving alone, as part of a larger complex, or are bound to large cellular components such as the membrane or chromosomal DNA. A remaining challenge has been to retrieve quantitative kinetic models, especially for molecules that rapidly switch between different diffusional states. Here, we present analytical diffusion distribution analysis (anaDDA), a framework that allows for extracting transition rates from distributions of apparent diffusion coefficients calculated from short trajectories that feature less than 10 localizations per track. Under the assumption that the system is Markovian and diffusion is purely Brownian, we show that theoretically predicted distributions accurately match simulated distributions and that anaDDA outperforms existing methods to retrieve kinetics, especially in the fast regime of 0.1–10 transitions per imaging frame. AnaDDA does account for the effects of confinement and tracking window boundaries. Furthermore, we added the option to perform global fitting of data acquired at different frame times to allow complex models with multiple states to be fitted confidently. Previously, we have started to develop anaDDA to investigate the target search of CRISPR-Cas complexes. In this work, we have optimized the algorithms and reanalyzed experimental data of DNA polymerase I diffusing in live Escherichia coli. We found that long-lived DNA interaction by DNA polymerase are more abundant upon DNA damage, suggesting roles in DNA repair. We further revealed and quantified fast DNA probing interactions that last shorter than 10 ms. AnaDDA pushes the boundaries of the timescale of interactions that can be probed with single-particle tracking and is a mathematically rigorous framework that can be further expanded to extract detailed information about the behavior of biomolecules in living cells.     

Plant architectural responses in simultaneous maize/soybean strip intercropping do not lead to a yield advantage

Maize/soybean strip intercropping is a commonly used system throughout China with high crop yields at reduced nutrient input compared to sole maize. Maize is the taller crop, and due to its dominance in light capture over soybean in the intercrop, maize is expected to outperform maize in sole cropping. Conversely, soybean is the subordinate crop and intercropped soybean plants are expected to perform worse than sole soybean. Crop plants show plastic responses in plant architecture to their growing conditions to forage for light and avoid shading. There is little knowledge on plant architectural responses to growing conditions in simultaneous (non-relay) intercropping and their relationship to species yields. A two-year field experiment with two simultaneous maize/soybean intercropping systems with narrow and wide strips was conducted to characterise architectural traits of maize and soybean plants grown as intercrop and sole crops. Intercropped maize plants, especially those in border rows, had substantially greater leaf area, biomass and yield than maize plants in sole crops. Intercropped soybean plants, especially those in border rows, had lower leaf area, biomass and yield than sole soybean plants. Overall intercrop performance was similar to that of sole crops, with the land equivalent ratio (LER) being only slightly greater than one (1.03–1.08). Soybean displayed typical shade avoidance responses in the intercrop, such as greater internode elongation and changes in specific leaf area, but these responses could not overcome the consequences of the competition with the taller maize plants. Therefore, in contrast to relay intercrop systems, in the studied simultaneous maize/soybean system, plastic responses did not contribute to practically relevant increases in resource capture and yield at whole system (i.e., intercrop) level.     

Rapid and comprehensive evaluation of microalgal fatty acids via untargeted gas chromatography and time‐of‐flight mass spectrometry

Due to their high triacylglyceride content, microalgae are intensively investigated for bio‐economy and food applications. However, lipid analysis is a laborious task incorporating extraction, transesterification and typically gas chromatographic measurement. Co‐elution induces a significant risk of fatty acid misidentification and thus, additional purification steps like thin layer chromatography are needed. Contrary to database matching approaches, solely targeted analysis is facilitated as compound identification is driven by matching retention times or indices with standard substances. In this context, a rapid workflow for the analysis of algal fatty acids is presented. In‐situ transesterification was used to simplify sample preparation and conditions were optimized towards fast processing. If results are needed at the very day of sampling, direct processing without a preceding drying step is feasible to obtain a rough estimate about the occurrence of the major compounds. Coupling gas chromatography and time‐of‐flight mass spectrometry enables untargeted analysis. Unknown compounds may be identified by structural reconstruction of their respective fragmentation patterns and by database matching to routinely avoid mismatches by co‐elution of disturbing agents. The developed workflow was successfully applied to derive the exact stereochemistry of all fatty acids from Chlorella vulgaris and a systematic shift depending on physiological state of the cells was confirmed.     

Physiological responses of Aureoumbra lagunensis and Synechococcus sp. to nitrogen addition in a mesocosm experiment

Aureoumbra lagunensis is the causative organism of the Texas brown tide and is notable because it dominated the Laguna Madre ecosystem from 1990 to 1997. This species is unusual because it has the highest known critical nitrogen to phosphorus ratio (N:P) for any microalgae ranging from 115 to 260, far higher than the 16N:1P Redfield ratio. Because of its high N:P ratio, Aureoumbra should be expected to respond to N additions that would not stimulate the growth of competitors having the Redfield ratio. To evaluate this prediction, a mesocosm experiment was performed in the Laguna Madre, a South Texas coastal lagoon, in which a mixed Aureoumbra–Synechococcus (a cyanobacterium) community was enclosed in 12 mesocosms and subjected to nitrogen addition (6 controls, 6 added ammonium) for 16 days. After day 4, added nitrogen did not significantly increase Aureoumbra specific growth rate but the alga retained dominance throughout the experiment (64–75% of total cell biovolume). In control mesocosms, Aureoumbra became less abundant during the first 4 days of the experiment but rebounded by the end of the experiment and was dominant over Synechococcus. Despite the lack of a strong positive growth response, Aureoumbra did respond physiologically to N addition. By the end of the experiment, the average N:P ratio of the Aureoumbra-dominated community was 86 in the N+ treatment and 41 in the control, indicating that the alga became less N-limited in the N+ treatment. The average C:N ratio was 6.6 in the N+ treatment (8.6 in the control) and suggests that the alga was not N-limited, however, C:N ratio may not be a good indicator of nitrogen limitation since this alga can produce significant quantities of carbon-containing extracellular polysaccharides, depending on growth conditions. Both Aureoumbra cellular chlorophyll fluorescence and cell size increased in response to added N, indicating a reduction in N limitation. It appeared that the N additions were not large and/or frequent enough to stimulate Aureoumbra growth. The main competitor, the unicellular cyanobacterium Synechococcus, responded positively to the nitrogen addition by increased specific growth rate. Unlike Aureoumbra, no significant effect on Synechococcus cellular pigment fluorescence or cell size was noted. Literature data suggest that Synechococcus, like Aureoumbra, may have a critical N:P ratio much higher than 16:1, which could explain its response.     

Ecological significance of light quality in optimizing plant defence

Plants balance the allocation of resources between growth and defence to optimize fitness in a competitive environment. Perception of neighbour‐detection cues, such as a low ratio of red to far‐red (R:FR) radiation, activates a suite of shade‐avoidance responses that include stem elongation and upward leaf movement, whilst simultaneously downregulating defence. This downregulation is hypothesized to benefit the plant either by mediating the growth‐defence balance in favour of growth in high plant densities or, alternatively, by mediating defence of individual leaves such that those most photosynthetically productive are best protected. To test these hypotheses, we used a 3D functional–structural plant model of Brassica nigra that mechanistically simulates the interactions between plant architecture, herbivory, and the light environment. Our results show that plant‐level defence expression is a strong determinant of plant fitness and that leaf‐level defence mediation by R:FR can provide a fitness benefit in high densities. However, optimal plant‐level defence expression does not decrease monotonically with plant density, indicating that R:FR mediation of defence alone is not enough to optimize defence between densities. Therefore, assessing the ecological significance of R:FR‐mediated defence is paramount to better understand the evolution of this physiological linkage and its implications for crop breeding.     

Effects of mesozooplankton removal and ammonium addition on planktonic trophic structure during a bloom of the Texas 'brown tide': a mesocosm study

A bloom of the alga Aureoumbra lagunensis, known as the Texas‘brown tide’, persisted in the Laguna Madre of Texasfor most of the 1990s. The dominant mesozooplankter in LagunaMadre, Acartia tonsa, does not feed on A. lagunensis, and duringblooms there are few other suitably sized phytoplankton cellsavailable to feed on. We hypothesized that these copepods increasedtheir feeding on microzooplankton, thereby reducing grazingpressure by microzooplankton on A. lagunensis and contributingto the persistence of this bloom. A mesocosm experiment wascarried out to test this hypothesis during the summer of 1999.Twelve fiberglass corral-type mesocosms were deployed in thefield for 16 days, each enclosing 1.2 m³ of Laguna Madre waterand 1.1 m² of natural benthos. Mesozooplankton were removedfrom six mesocosms with a 153 µm mesh dip net every 4days; the other six mesocosms were treated in the same way,except that the contents of the net were returned to the mesocosm.For each zooplankton treatment, half of the mesocosms were dosedwith 40 µm NH₄ at 4 day intervals, and half received noadditions. Phytoplankton populations in these mesocosms at thestart of the experiment were dominated by A. lagunensis andthe cyanobacterium Synechococcus spp. Growth rates of A. lagunensiswere higher in mesocosms without ammonium additions, providingno evidence for nitrogen limitation. Acartia tonsa populationswere reduced by 50% in the zooplankton removal mesocosms, andciliate populations were significantly higher. The increasein ciliate population had no significant impact on A. lagunensispopulation dynamics, however, providing no evidence to supportthe hypothesis that a trophic cascade reducing microzooplanktonpopulations contributed to the persistence of the brown-tidebloom. In contrast, populations of Synechococcus sp. showedevidence of both ‘top–down’ and ‘bottom–up’control; they grew faster in nutrient addition mesocosms andhad lower populations in mesocosms with increased densitiesof ciliate grazers.     

Cytometric quantification of nitrate reductase by immunolabeling in the marine diatom Skeletonema costatum

BACKGROUND: The uptake of nitrate by phytoplankton is a central issue in biological oceanography due to its importance to primary production and vertical flux of biogenic carbon. Nitrate reductase catalyzes the first step of nitrate assimilation, the reduction of NO(3) to NO(2). A cytometric protocol to detect and quantify relative changes in nitrate reductase (NR) protein content of the marine centric diatom Skeletonema costatum is presented. METHODS: Immunolabeling of NR protein was achieved with polyclonal antibodies raised against S.costatum NR. Antisera specific to a NR protein subunit and to a NR polypeptide sequence were compared, and cytometric results of NR protein abundance were related to Western analyses. Changes in cellular NR abundance and activity were followed during an upwelling simulation experiment in which S. costatum was exposed to a shift from ammonia to nitrate as major nitrogen source. RESULTS: NR protein could be detected in NO(3)-grown cells and at extremely low levels hardly discernible by Western Blot densiometry in NH(4)-grown cells. The protocol allowed observation of early stages of NR induction during an upwelling simulation. NR abundance increased after the nutrient shift to reach a new physiological "steady-state" 96 hrs later. NR activity exhibited diel variation with maxima at mid-day. NR abundance as estimated by both flow cytometry and Western analysis exhibited a hyperbolic relationship to NR activity. This pattern suggests post-translational activation of NR protein. CONCLUSIONS: The presented protocol allows the differentiation of NH(4)- versus NO(3)-grown algae as well as the monitoring of early stages in the induction of nitrate assimilatory capacities.     

Lipotoxicity and steatohepatitis in an overfed mouse model for non-alcoholic fatty liver disease

The major risk factors for non-alcoholic fatty liver disease (NAFLD) are obesity, insulin resistance and dyslipidemia. The cause for progression from the steatosis stage to the inflammatory condition (non-alcoholic steatohepatitis (NASH)) remains elusive at present. Aim of this study was to test whether the different stages of NAFLD as well as the associated metabolic abnormalities can be recreated in time in an overfed mouse model and study the mechanisms underlying the transition from steatosis to NASH.Male C57Bl/6J mice were subjected to continuous intragastric overfeeding with a high-fat liquid diet (HFLD) for different time periods. Mice fed a solid high-fat diet (HFD) ad libitum served as controls. Liver histology and metabolic characteristics of liver, white adipose tisue (WAT) and plasma were studied.Both HFD-fed and HFLD-overfed mice initially developed liver steatosis, but only the latter progressed in time to NASH. NASH coincided with obesity, hyperinsulinemia, loss of liver glycogen and hepatic endoplasmatic reticulum stress. Peroxisome proliferator-activated receptor γ (Pparγ), fibroblast growth factor 21 (Fgf21), fatty acid binding protein (Fabp) and fatty acid translocase (CD36) were induced exclusively in the livers of the HFLD-overfed mice. Inflammation, reduced adiponectin expression and altered expression of genes that influence adipogenic capacity were only observed in WAT of HFLD-overfed mice.In conclusion: this dietary mouse model displays the different stages and the metabolic settings often found in human NAFLD. Lipotoxicity due to compromised adipose tissue function is likely associated with the progression to NASH, but whether this is cause or consequence remains to be established.     

Paraoxonase-1 is a major determinant of clopidogrel efficacy

Clinical efficacy of the antiplatelet drug clopidogrel is hampered by its variable biotransformation into the active metabolite. The variability in the clinical response to clopidogrel treatment has been attributed to genetic factors, but the specific genes and mechanisms underlying clopidogrel bioactivation remain unclear. Using in vitro metabolomic profiling techniques, we identified paraoxonase-1 (PON1) as the crucial enzyme for clopidogrel bioactivation, with its common Q192R polymorphism determining the rate of active metabolite formation. We tested the clinical relevance of the PON1 Q192R genotype in a population of individuals with coronary artery disease who underwent stent implantation and received clopidogrel therapy. PON1 QQ192 homozygous individuals showed a considerably higher risk than RR192 homozygous individuals of stent thrombosis, lower PON1 plasma activity, lower plasma concentrations of active metabolite and lower platelet inhibition. Thus, we identified PON1 as a key factor for the bioactivation and clinical activity of clopidogrel. These findings have therapeutic implications and may be exploited to prospectively assess the clinical efficacy of clopidogrel.