Quantifying Biomass Changes of Single CD8+ T Cells during Antigen Specific Cytotoxicity

Existing approaches that quantify cytotoxic T cell responses rely on bulk or surrogate measurements which impede the direct identification of single activated T cells of interest. Single cell microscopy or flow cytometry methodologies typically rely on fluorescent labeling, which limits applicability to primary cells such as human derived T lymphocytes. Here, we introduce a quantitative method to track single T lymphocyte mediated cytotoxic events within a mixed population of cells using live cell interferometry (LCI), a label-free microscopy technique that maintains cell viability. LCI quantifies the mass distribution within individual cells by measuring the phase shift caused by the interaction of light with intracellular biomass. Using LCI, we imaged cytotoxic T cells killing cognate target cells. In addition to a characteristic target cell mass decrease of 20–60% over 1–4 h following attack by a T cell, there was a significant 4-fold increase in T cell mass accumulation rate at the start of the cytotoxic event and a 2–3 fold increase in T cell mass relative to the mass of unresponsive T cells. Direct, label-free measurement of CD8+ T and target cell mass changes provides a kinetic, quantitative assessment of T cell activation and a relatively rapid approach to identify specific, activated patient-derived T cells for applications in cancer immunotherapy.     

An Arabidopsis lipid map reveals differences between tissues and dynamic changes throughout development

Mass spectrometry is the predominant analytical tool used in the field of plant lipidomics. However, there are many challenges associated with the mass spectrometric detection and identification of lipids because of the highly complex nature of plant lipids. Studies into lipid biosynthetic pathways, gene functions in lipid metabolism, lipid changes during plant growth and development, and the holistic examination of the role of plant lipids in environmental stress responses are often hindered. Here, we leveraged a robust pipeline that we previously established to extract and analyze lipid profiles of different tissues and developmental stages from the model plant Arabidopsis thaliana. We analyzed seven tissues at several different developmental stages and identified more than 200 lipids from each tissue analyzed. The data were used to create a web-accessible in silico lipid map that has been integrated into an electronic Fluorescent Pictograph (eFP) browser. This in silico library of Arabidopsis lipids allows the visualization and exploration of the distribution and changes of lipid levels across selected developmental stages. Furthermore, it provides information on the characteristic fragments of lipids and adducts observed in the mass spectrometer and their retention times, which can be used for lipid identification. The Arabidopsis tissue lipid map can be accessed at http://bar.utoronto.ca/efp_arabidopsis_lipid/cgi-bin/efpWeb.cgi .     

Circadian clock adjustment to plant iron status depends on chloroplast and phytochrome function

Plant chloroplasts are not only the main cellular location for storage of elemental iron (Fe), but also the main site for Fe, which is incorporated into chlorophyll, haem and the photosynthetic machinery. How plants measure internal Fe levels is unknown. We describe here a new Fe‐dependent response, a change in the period of the circadian clock. In Arabidopsis, the period lengthens when Fe becomes limiting, and gradually shortens as external Fe levels increase. Etiolated seedlings or light‐grown plants treated with plastid translation inhibitors do not respond to changes in Fe supply, pointing to developed chloroplasts as central hubs for circadian Fe sensing. Phytochrome‐deficient mutants maintain a short period even under Fe deficiency, stressing the role of early light signalling in coupling the clock to Fe responses. Further mutant and pharmacological analyses suggest that known players in plastid‐to‐nucleus signalling do not directly participate in Fe sensing. We propose that the sensor governing circadian Fe responses defines a new retrograde pathway that involves a plastid‐encoded protein that depends on phytochromes and the functional state of chloroplasts.     

The oxidation-reduction potential of P-700 in chloroplast lamellae and subchloroplast particles

The oxidation-reduction potential of P-700 has been determined in chloroplast lamellae and in subchloroplast particles by measuring the magnitude of flash-induced absorption changes at 820 or at 703 nm (due to the oxidation of P-700) in the presence of known concentrations of potassium ferro- and ferricyanide. A midpoint potential of about +490 mV was determined in chloroplast lamellae and in particles prepared with digitonin (D-144) or Triton (TSF-1). A lower potential was determined with Photosystem I particles obtained after harsher treatments with Triton or a mixture of detergents. The potential is even lower in chlorophyll-protein complex I particles prepared with sodium dodecyl sulfate (about +430 mV). Very similar values were determined from oxidized minus reduced spectra measured with a double-beam spectrophotometer. Titrations were also made with D-144 and TSF-I particles, with 66% glyeerol in the buffer, at 21 °C and at 77 °K. P-700 was found to be half-oxidized at ferricyanide/ferrocyanide ratios of about 60 at 21 °C and of about 1 at 77 °K. This shows that the redox equilibrium is largely perturbed by the cooling process.     

Queuine in plants and plant tRNAs: Differences between embryonic tissue and mature leaves

In eubacterial and eukaryotic tRNAs specific for Asn, Asp, His and Tyr the modified deazaguanosinederivative queuosine occurs in position 34, the first position of the anticodon. Analysis of unfractionated tRNAs from wheat and from tobacco leaves shows that these tRNAs contain high amounts of guanosine (G) in place of queuosine (Q). This was measured by the exchange of G34 for [³H]guanine catalysed by the specific tRNA guanine transglycosylase from E. coli. Upon gel electrophoretic separation of the labeled tRNAs, seven Q-deficient tRNA species including isoacceptors are detectable. Two are identified as cytoplasmic tRNAs^Tyr and tRNA^Asp and two represent chloroplast tRNA^Tyr isoacceptors. In contrast to leaf cytoplasm and chloroplasts, wheat germ has low amounts of tRNAs with G34 in place of Q.A new enzymatic assay is described for quantitation of free queuine in cells and tissues. Analysis of queuine in plant tissues shows that wheat germ contains about 200 ng queuine per g wet weight. In wheat and tobacco leaves queuine is present, if at all, in amounts lower than 10 ng/g wet weight. The absence of Q in tRNAs from plant leaves is therefore caused by a deficiency of queuine. Tobacco cells cultivated in a synthetic medium without added queuine do not contain Q in tRNA, indicating that these rapidly growing cells do not synthesize queuine de novo.     

Gibberellic Acid effects on greening in pea seedlings

The effect of gibberellic acid (GA) on light-induced greening of etiolated pea plants (Pisum sativum [L.] cultivars Alaska and Progress) was characterized. Progress, a GA-deficient dwarf of Alaska, was found to accumulate chlorophyll and light harvesting chlorophyll protein associated with photosystem II (LHC-II) more rapidly than Alaska, Alaska treated with GA, or Progress treated with GA. A slightly lower chlorophyll content was noted after 24 hours of light induced greening for Alaska treated with GA relative to untreated Alaska. GA-treated Progress, Alaska, and GA-treated Alaska all gave essentially identical patterns for LHC-II accumulation. Similar patterns of LHC-II mRNA induction were found in all four treatments indicating that differences in mRNA induction did not cause differences in LHC-II accumulation. Chlorophyll and LHC-II accumulation in each treatment followed the same patterns of accumulation and a significant correlation (at the 0.01 level of significance) was found between chlorophyll and LHC-II content. Since Progress treated with GA accumulated LHC-II and chlorophyll in a manner similar to that of Alaska, it is clear that GA alters the process of greening either directly or indirectly.     

Molecular characterization of the diurnal/circadian expression of the chlorophyll a/b-binding proteins in leaves of tomato and other dicotyledonous and monocotyledonous plant species

Diurnal oscillations of steady-state mRNA levels encoding the chlorophyll a/b-binding proteins were monitored inLycopersicon esculentum, Glycine max, Phaseolus vulgaris, P. aureus, P. coccineus, Pisum sativum, Sinapis alba, Hordeum vulgare, Triticum aestivum andZea mays. In these plant speciescab mRNA accumulation increases and decreases periodically indicating i) that the expression of the genes for chlorophyll a/b-binding proteins (cab genes) is controlled by a circadian rhythm, and ii) that the rhythm is widely distributed among monocotyledonous and dicotyledonous plant species. A detailed characterization of the pattern ofcab mRNA expression in tomato leaves shows that the amplitude of the oscillation is dependent on i) the developmental stage of the leaves, ii) the circadian phase and duration of light and iii) the circadian phase and duration of darkness. In addition to the chlorophyll a/b-binding proteins, genes coding for other cellular functions were examined for cyclic variations of their mRNA levels. The analysis includes genes involved in i) carbon metabolism (e.g. phosphoenolpyruvate carboxylase, pyruvate orthophosphate dikinase, alpha amylase, fructose-1,6-bisphosphate aldolase, triosephosphate isomerase), ii) photosynthesis (large and small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, Q_B-binding protein, reaction-center protein of photosystem I) and iii) other physiological or morphological reactions (e.g. ubiquitin, actin). However, no periodic fluctuation pattern was detected for the mRNA levels of these genes in tomato and maize leaves.     

An empirical analysis of the factors contributing to 20-year decrease in soil pH in an old-field plantation of loblolly pine

The pH of weak-acid solutions is controlled by acid concentration (HA + A^–), the degree of acid dissociation (A^–/HA), and the strength of the acids present (pK_a). We developed an empirical approach that allows the relative importance of each of these factors to be estimated for soils. This empirical model was applied to soils collected from an old-field plantation of loblolly pine (Pinus taeda L.) at 5 and 25 years of age. During this period, soil pH dropped by 0.3 to 0.8 units, and extractable calcium, magnesium and potassium declined by 20 to 80%. The empirical model indicates that the decline in pH resulted largely from the reduction in base saturation of the exchange complex. However, the average acid strength of the exchange complex decreased during the 20 years, preventing a greater decline of perhaps 0.1 to 0.2 units in the observed pH. The rate of decrease in the acid neutralizing capacity to pH 3.5 was about 1.3 kmol_c/ha annually, while the increase in base neutralizing capacity was about 2.7 and 1.6 kmol_c/ha annually to pH 5.5 and 8.2, respectively. Extractable alkali and alkaline earth cations declined by about 2.2 kmol_c/ha annually, matched by the rate of increase in aluminium. These changes demonstrated the dynamic nature of poorly buffered soils, and indicated that changes in soil acidity may be expected over a period of decades (especially following changes in land-use).