Structure of the light harvesting antenna from Rhodospirillum molischianum studied by electron microscopy

The structure of two types of isolated light-harvesting antenna complexes from Rhodospirillum molischianum was studied by electron microscopy and image analysis. The B870 reaction center complex forms an almost circular particle with a diameter in the plane of the membrane of about 10.7-11.2 nm. A complex consists of a reaction center surrounded most likely by 12 B870 antennae units. An asymmetrical protrusion, consisting of the cytochrome subunit, gives the reaction center a height of 11.5 nm. The peripheral B800-820 complex is cylindrical, with a diameter of 5.3 nm and a small central indentation. At low resolution, it is structurally very similar to the B800-850 complex of Rhodobacter sphaeroides and is considered to be most likely of the α6β6 type.

Electron Microscopic Structural Analysis of Photosystem I, Photosystem II, and the Cytochrome b6/f Complex from Green Plants and Cyanobacteria

Electron microscopy (EM) in combination with image analysis is a powerful technique to study protein structure at low- and high resolution. Since electron micrographs of biological objects are very noisy, substantial improvement of image quality can be obtained by averaging individual projections. Crystallographic and noncrystallographic averaging methods are available and have been applied to study projections of the large protein complexes embedded in photosynthetic membranes from cyanobacteria and higher plants. Results of EM on monomeric and trimeric Photosystem I complexes, on monomeric and dimeric Photosystem II complexes, and on the monomeric cytochrome b6/f complex are discussed.

Isolation and Structural Characterization of Monomeric and Trimeric Photosystem I Complexes (P700·FA/FB and P700·Fx) from the Cyanobacterium Synechocystis PCC 6803

An isolation procedure was developed for the cyanobacterium Synechocystis 6803 (and 6714) which yields both monomeric and trimeric photosystem I complexes (P700·FA/FB complexes) depleted of the stroma-exposed subunits PsaC, -D, and -E (P700·FX complexes). Analysis by high resolution gel electrophoresis in combination with immunoblotting and N-terminal sequencing reveals the selective and quantitative removal of PsaC, -D, and -E from the P700·FA/FB complex, containing PsaA, -B, -C, -D, -E, -F, -K, -L and at least two subunits ≤4 kDa. Monomeric and trimeric P700·Fx complexes show an identical subunit composition and an identical charge recombination half-time of 750 ± 250 µs as determined by flash-induced absorption change measurements, reflecting the quantitative loss of iron-sulfur clusters FA/FB and the presence of cluster FX. The existence of a stable trimeric P700·FX complex enables a detailed structural analysis by electron microscopy with high resolution. Comparison of averaged top and side view projections of P700·FX and P700·FA/FB complexes show that the height of the complex is reduced by about 2.5-3.3 nm upon removal of the three stroma-exposed subunits and indicate the position of these three subunits on the PS I surface. While the outer contours of the stroma exposed mass of PS I agree very well with the three-dimensional crystal analysis recently published for trimeric PS I of Synechococcus elongatus, only the structural analysis presented here is able to assign the stroma-exposed mass exclusively to the subunits PsaC, -D, and -E and to exclude a contribution of other subunits.

Structural characterization of the B800-850 and B875 light-harvesting antenna complexes from Rhodobacter sphaeroides by electron microscopy

The structure and aggregation behavior of B800-850 (LHII) and B875 (LHI) antenna complexes of Rhodobacter sphaeroides were studied by electron microscopy. Single molecular projections (top views and side views) of isolated particles were analyzed. The B800-850 complexes, isolated as 150 kDa particles, are cylindrical with a diameter of 8.5 nm and with a height of 6.5 nm. If corrected for attached detergent, the actual diameter in the plane of the membrane would be about 5.1 nm. Stain accumulation in the center of the structure indicates a small indentation, therefore a ring-shaped structure is expected. The size, shape and estimated mass give evidence for a B800-850 complex structure consisting of 4-6 αβ heterodimers, if in an αnβn ring-shaped configuration. The B875 complexes, isolated as 360 kDa particles, are dimeric units, but the two monomers are only loosely connected. A single B875 unit has a corrected diameter of about 5.2 nm and a height of 6.2 nm, similar to the B800-850 unit. Therefore, the B875 complex most likely has the same αnβn configuration. Image analysis of B800-850 projections points to a 3- or 6-fold symmetry in the top views, rather than a 4- or 5-fold symmetry. Therefore, our results are most compatible with an α6β6 configuration.

DeltaScan for the Assessment of Acute Encephalopathy and Delirium in ICU and non-ICU Patients, a Prospective Cross-Sectional Multicenter Validation Study

Objectives: To measure the diagnostic accuracy of DeltaScan: a portable real-time brain state monitor for identifying delirium, a manifestation of acute encephalopathy (AE) detectable by polymorphic delta activity (PDA) in single-channel electroencephalograms (EEGs). Design: Prospective cross-sectional study. Setting: Six Intensive Care Units (ICU's) and 17 non-ICU departments, including a psychiatric department across 10 Dutch hospitals. Participants: 494 patients, median age 75 (IQR:64-87), 53% male, 46% in ICUs, 29% delirious. Measurements: DeltaScan recorded 4-minute EEGs, using an algorithm to select the first 96 seconds of artifact-free data for PDA detection. This algorithm was trained and calibrated on two independent datasets. Methods: Initial validation of the algorithm for AE involved comparing its output with an expert EEG panel's visual inspection. The primary objective was to assess DeltaScan's accuracy in identifying delirium against a delirium expert panel's consensus. Results: DeltaScan had a 99% success rate, rejecting 6 of the 494 EEG's due to artifacts. Performance showed and an Area Under the Receiver Operating Characteristic Curve (AUC) of 0.86 (95% CI: 0.83-0.90) for AE (sensitivity: 0.75, 95%CI=0.68-0.81, specificity: 0.87 95%CI=0.83-0.91. The AUC was 0.71 for delirium (95%CI=0.66-0.75, sensitivity: 0.61 95%CI=0.52-0.69, specificity: 72, 95%CI=0.67-0.77). Our validation aim was an NPV for delirium above 0.80 which proved to be 0.82 (95%CI: 0.77-0.86). Among 84 non-delirious psychiatric patients, DeltaScan differentiated delirium from other disorders with a 94% (95%CI: 87-98%) specificity. Conclusions: DeltaScan can diagnose AE at bedside and shows a clear relationship with clinical delirium. Further research is required to explore its role in predicting delirium-related outcomes.</p

DeltaScan for the Assessment of Acute Encephalopathy and Delirium in ICU and non-ICU Patients, a Prospective Cross-Sectional Multicenter Validation Study

Objectives: To measure the diagnostic accuracy of DeltaScan: a portable real-time brain state monitor for identifying delirium, a manifestation of acute encephalopathy (AE) detectable by polymorphic delta activity (PDA) in single-channel electroencephalograms (EEGs). Design: Prospective cross-sectional study. Setting: Six Intensive Care Units (ICU's) and 17 non-ICU departments, including a psychiatric department across 10 Dutch hospitals. Participants: 494 patients, median age 75 (IQR:64-87), 53% male, 46% in ICUs, 29% delirious. Measurements: DeltaScan recorded 4-minute EEGs, using an algorithm to select the first 96 seconds of artifact-free data for PDA detection. This algorithm was trained and calibrated on two independent datasets. Methods: Initial validation of the algorithm for AE involved comparing its output with an expert EEG panel's visual inspection. The primary objective was to assess DeltaScan's accuracy in identifying delirium against a delirium expert panel's consensus. Results: DeltaScan had a 99% success rate, rejecting 6 of the 494 EEG's due to artifacts. Performance showed and an Area Under the Receiver Operating Characteristic Curve (AUC) of 0.86 (95% CI: 0.83-0.90) for AE (sensitivity: 0.75, 95%CI=0.68-0.81, specificity: 0.87 95%CI=0.83-0.91. The AUC was 0.71 for delirium (95%CI=0.66-0.75, sensitivity: 0.61 95%CI=0.52-0.69, specificity: 72, 95%CI=0.67-0.77). Our validation aim was an NPV for delirium above 0.80 which proved to be 0.82 (95%CI: 0.77-0.86). Among 84 non-delirious psychiatric patients, DeltaScan differentiated delirium from other disorders with a 94% (95%CI: 87-98%) specificity. Conclusions: DeltaScan can diagnose AE at bedside and shows a clear relationship with clinical delirium. Further research is required to explore its role in predicting delirium-related outcomes.</p