Protection of photosystem II against damage from excess light by nonphotochemical quenching (NPQ) includes responses on a wide range of timescales. The onset of the various phases of NPQ overlap in time making it difficult to discern if they influence each other or involve different photophysical mechanisms. To unravel the complex relationship of the known actors in NPQ, we perform fluorescence lifetime snapshot measurements throughout multiple cycles of alternating 2 min periods of high light and darkness. By comparing the data with an empirically based mathematical model that describes both fast and slow quenching responses, we suggest that the rapidly reversible quenching response depends on the state of the slower response. By studying a series of Arabidopsis thaliana mutants, we find that removing zeaxanthin (Zea) or enhancing PsbS concentration, for example, influences the amplitudes of the slow quenching induction and recovery, but not the timescales. The plants' immediate response to high light appears independent of the illumination history, while PsbS and Zea have distinct roles in both quenching and recovery. We further identify two parameters in our model that predominately influence the recovery amplitude and propose that our approach may prove useful for screening new mutants or overexpressors with enhanced biomass yields under field conditions.
<p>We explore the photoprotection dynamics of Nannochloropsis oceanica using time-correlated single photon counting under regular and irregular actinic light sequences. The varying light sequences mimic natural conditions, allowing us to probe the real-time response of non-photochemical quenching (NPQ) pathways. Durations of fluctuating light exposure during a fixed total experimental time and prior light exposure of the algae are both found to have a profound effect on NPQ. These observations are rationalized with a quantitative model based on the xanthophyll cycle and the protonation of LHCX1. The model is able to accurately describe the dynamics of non-photochemical quenching across a variety of light sequences. The combined model and observations suggest that the accumulation of a quenching complex, likely zeaxanthin bound to a protonated LHCX1, is responsible for the gradual rise in NPQ. Additionally, the model makes specific predictions for the light sequence dependence of xanthophyll concentrations that are in reasonable agreement with independent chromatography measurements taken during a specific light/dark sequence.
ObjectivesTo determine if spatial variations in gut permeability play a role in regulating type 1 diabetes (T1D) progression.Research design and methodsSpatially resolved duodenum, jejunum, ileum, and large intestine sections from end-stage T1D non-obese diabetic (NOD) mice were probed by immunohistochemistry to quantify zonulin levels as a measure of gut permeability in early-progressor and late-progressor NOD mice in comparison with non-progressor NOD mice and healthy NOR/LtJ control mice.ResultsZonulin levels were elevated in the small and large intestines in early-progressor and late-progressor NOD mice in comparison with non-progressor NOD mice and healthy NOR control mice. In early-onset mice, elevated zonulin levels were maximum in the duodenum and jejunum and decreased in the ileum and large intestine. In late-progressor mice, zonulin levels were elevated almost evenly along the small and large intestines. In non-progressor NOD mice, zonulin levels were comparable with NOR control levels in both the small and large intestines.ConclusionsElevated zonulin expression levels indicated that gut permeability was increased both in the small and large intestines in NOD mice that progressed to end-stage T1D in comparison with non-progressor NOD mice and healthy NOR control mice. Highest elevations in zonulin levels were observed in the duodenum and jejunum followed by the ileum and large intestines. Spatial variations in gut permeability appeared to play a role in regulating the rate and severity of T1D progression in NOD mice indicating that spatial variations in gut permeability should be investigated as a potentially important factor in human T1D progression.
Photosynthetic organisms use sunlight as the primary energy source to fix CO 2 .However, in nature, light energy is highly variable, reaching levels of saturation for periods ranging from milliseconds to hours. In the green microalga Chlamydomonas reinhardtii, safe dissipation of excess light energy by nonphotochemical quenching (NPQ) is mediated by light-harvesting complex stressrelated (LHCSR) proteins and redistribution of light-harvesting antennae between the photosystems (state transition). Although each component
Photosynthetic organisms use sunlight as the primary energy source to fix CO2. However, in the environment, light energy fluctuates rapidly and often exceeds saturating levels for periods ranging from seconds to hours, which can lead to detrimental effects for cells. Safe dissipation of excess light energy occurs primarily by non-photochemical quenching (NPQ) processes. In the model green microalga Chlamydomonas reinhardtii, photoprotective NPQ is mostly mediated by pH-sensing light-harvesting complex stress-related (LHCSR) proteins and the redistribution of light-harvesting antenna proteins between the photosystems (state transition). Although each component underlying NPQ has been documented, their relative contributions to the dynamic functioning of NPQ under fluctuating light conditions remains unknown. Here, by monitoring NPQ throughout multiple high light-dark cycles with fluctuation periods ranging from 1 to 10 minutes, we show that the dynamics of NPQ depend on the frequency of light fluctuations. Mutants impaired in the accumulation of LHCSRs (npq4, lhcsr1, and npq4lhcsr1) showed significantly less quenching during illumination, demonstrating that LHCSR proteins are responsible for the majority of NPQ during repetitive exposure to high light fluctuations. Activation of NPQ was also observed during the dark phases of light fluctuations, and this was exacerbated in mutants lacking LHCSRs. By analyzing 77K chlorophyll fluorescence spectra and chlorophyll fluorescence lifetimes and yields in a mutant impaired in state transition, we show that this phenomenon arises from state transition. Finally, we quantified the contributions of LHCSRs and state transition to the overall NPQ amplitude and dynamics for all light periods tested and compared those with cell growth under various periods of fluctuating light. These results highlight the dynamic functioning of photoprotection under light fluctuations and open a new way to systematically characterize the photosynthetic response to an ever-changing light environment.
High throughput (HTCD) using 96 and 384 multiplate for the optimisation of protein crystallization conditions, protein-ligand binding screening, and CD Imaging (CDi) of thin films of materials (proteins, nucleic acids, carbohydrates, polymers with embedded drugs, organic LED, and in general any chiral material with or without achiral substrates) are the results of the unique highly collimated microbeam light generated at B23 beamline at Diamond Light Source in the vacuum UV to visible wavelength range (130-650nm). B23 is the only bespoke beamline worldwide to characterise the structural and conformational properties of biologically important molecules using micro-devices unattainable with bespoke bench-top CD instruments. Recent applications and latest beamline upgrades will be discussed.
2812-PosWidefield Multi-Frequency Fluorescence Lifetimeimaging using a Two-Tap CMOS Camera Withlateral Electric Field Charge Modulators Widefield frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) measures fluorescence lifetime of entire images in a fast and efficient manner. We report a widefield FD-FLIM system based on a CMOS camera equipped with two-tap true correlated double sampling (CDS) lock-in pixels and lateral electric field charge modulators. Due to the fast intrinsic response and modulation of the camera, our system allows parallel multi-frequency FLIM in one measurement via Fast Fourier Transform. We demonstrate that at a fundamental frequency of 20MHz, 31-harmonics (620MHz) can be measured with 64 phase images per laser repetition period. As a proof of principle, we analyzed cells transfected with Cerulean and with a construct of Cerulean-Venus that shows Förster resonance energy transfer at different modulation frequencies. We also tracked the temperature change of living cells via the fluorescence lifetime of Rhodamine B at different frequencies. Furthermore, we can rapidly distinguish the pre-implantation embryo development stages at the optimal frequency by the fluorescence lifetime differences of their intrinsic fluorescence in 2D phasor plots. These results indicate that our widefield multi-frequency FD-FLIM system is a valuable tool in the biomedical field. [Work done in part with funds from NIH P41-GM103540].
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