Augmenting light coverage for photosynthesis through YFP-enhanced charge separation at the Rhodobacter sphaeroides reaction centre.

Photosynthesis uses a limited range of the solar spectrum, so enhancing spectral coverage could improve the efficiency of light capture. Here, we show that a hybrid reaction centre (RC)/yellow fluorescent protein (YFP) complex accelerates photosynthetic growth in the bacterium Rhodobacter sphaeroides. The structure of the RC/YFP-light-harvesting 1 (LH1) complex shows the position of YFP attachment to the RC-H subunit, on the cytoplasmic side of the RC complex. Fluorescence lifetime microscopy of whole cells and ultrafast transient absorption spectroscopy of purified RC/YFP complexes show that the YFP-RC intermolecular distance and spectral overlap between the emission of YFP and the visible-region (QX) absorption bands of the RC allow energy transfer via a Förster mechanism, with an efficiency of 40±10%. This proof-of-principle study demonstrates the feasibility of increasing spectral coverage for harvesting light using non-native genetically-encoded light-absorbers, thereby augmenting energy transfer and trapping in photosynthesis

Engineering of B800 bacteriochlorophyll binding site specificity in the Rhodobacter sphaeroides LH2 antenna.

The light-harvesting 2 complex (LH2) of the purple phototrophic bacterium Rhodobacter sphaeroides is a highly efficient, light-harvesting antenna that allows growth under a wide-range of light intensities. In order to expand the spectral range of this antenna complex, we first used a series of competition assays to measure the capacity of the non-native pigments 3-acetyl chlorophyll (Chl) a, Chl d, Chl f or bacteriochlorophyll (BChl) b to replace native BChl a in the B800 binding site of LH2. We then adjusted the B800 site and systematically assessed the binding of non-native pigments. We find that Arg-10 of the LH2 β polypeptide plays a crucial role in binding specificity, by providing a hydrogen-bond to the 3-acetyl group of native and non-native pigments. Reconstituted LH2 complexes harbouring the series of (B)Chls were examined by transient absorption and steady-state fluorescence spectroscopies. Although slowed 10-fold to ~6 ps, energy transfer from Chl a to B850 BChl a remained highly efficient. We measured faster energy-transfer time constants for Chl d (3.5 ps) and Chl f (2.7 ps), which have red-shifted absorption maxima compared to Chl a. BChl b, red-shifted from the native BChl a, gave extremely rapid (≤0.1 ps) transfer. These results show that modified LH2 complexes, combined with engineered (B)Chl biosynthesis pathways in vivo, have potential for retaining high efficiency whilst acquiring increased spectral range

Structures of Rhodopseudomonas palustris RC-LH1 complexes with open or closed quinone channels

The reaction-center light-harvesting complex 1 (RC-LH1) is the core photosynthetic component in purple phototrophic bacteria. We present two cryo–electron microscopy structures of RC-LH1 complexes from Rhodopseudomonas palustris. A 2.65-Å resolution structure of the RC-LH114-W complex consists of an open 14-subunit LH1 ring surrounding the RC interrupted by protein-W, whereas the complex without protein-W at 2.80-Å resolution comprises an RC completely encircled by a closed, 16-subunit LH1 ring. Comparison of these structures provides insights into quinone dynamics within RC-LH1 complexes, including a previously unidentified conformational change upon quinone binding at the RC QB site, and the locations of accessory quinone binding sites that aid their delivery to the RC. The structurally unique protein-W prevents LH1 ring closure, creating a channel for accelerated quinone/quinol exchange

Completion Dissection or Observation for Sentinel-Node Metastasis in Melanoma.

Sentinel-lymph-node biopsy is associated with increased melanoma-specific survival (i.e., survival until death from melanoma) among patients with node-positive intermediate-thickness melanomas (1.2 to 3.5 mm). The value of completion lymph-node dissection for patients with sentinel-node metastases is not clear. In an international trial, we randomly assigned patients with sentinel-node metastases detected by means of standard pathological assessment or a multimarker molecular assay to immediate completion lymph-node dissection (dissection group) or nodal observation with ultrasonography (observation group). The primary end point was melanoma-specific survival. Secondary end points included disease-free survival and the cumulative rate of nonsentinel-node metastasis. Immediate completion lymph-node dissection was not associated with increased melanoma-specific survival among 1934 patients with data that could be evaluated in an intention-to-treat analysis or among 1755 patients in the per-protocol analysis. In the per-protocol analysis, the mean (±SE) 3-year rate of melanoma-specific survival was similar in the dissection group and the observation group (86±1.3% and 86±1.2%, respectively; P=0.42 by the log-rank test) at a median follow-up of 43 months. The rate of disease-free survival was slightly higher in the dissection group than in the observation group (68±1.7% and 63±1.7%, respectively; P=0.05 by the log-rank test) at 3 years, based on an increased rate of disease control in the regional nodes at 3 years (92±1.0% vs. 77±1.5%; P<0.001 by the log-rank test); these results must be interpreted with caution. Nonsentinel-node metastases, identified in 11.5% of the patients in the dissection group, were a strong, independent prognostic factor for recurrence (hazard ratio, 1.78; P=0.005). Lymphedema was observed in 24.1% of the patients in the dissection group and in 6.3% of those in the observation group. Immediate completion lymph-node dissection increased the rate of regional disease control and provided prognostic information but did not increase melanoma-specific survival among patients with melanoma and sentinel-node metastases. (Funded by the National Cancer Institute and others; MSLT-II ClinicalTrials.gov number, NCT00297895 .)

Therapeutic Value of Sentinel Lymph Node Biopsy in Patients With Melanoma: A Randomized Clinical Trial.

Sentinel lymph node (SLN) biopsy is a standard staging procedure for cutaneous melanoma. Regional disease control is a clinically important therapeutic goal of surgical intervention, including nodal surgery. To determine how frequently SLN biopsy without completion lymph node dissection (CLND) results in long-term regional nodal disease control in patients with SLN metastases. The second Multicenter Selective Lymphadenectomy Trial (MSLT-II), a prospective multicenter randomized clinical trial, randomized participants with SLN metastases to either CLND or nodal observation. The current analysis examines observation patients with regard to regional nodal recurrence. Trial patients were aged 18 to 75 years with melanoma metastatic to SLN(s). Data were collected from December 2004 to April 2019, and data were analyzed from July 2020 to January 2022. Nodal observation with ultrasonography rather than CLND. In-basin nodal recurrence. Of 823 included patients, 479 (58.2%) were male, and the mean (SD) age was 52.8 (13.8) years. Among 855 observed basins, at 10 years, 80.2% (actuarial; 95% CI, 77-83) of basins were free of nodal recurrence. By univariable analysis, freedom from regional nodal recurrence was associated with age younger than 50 years (hazard ratio [HR], 0.49; 95% CI, 0.34-0.70; P < .001), nonulcerated melanoma (HR, 0.36; 95% CI, 0.36-0.49; P < .001), thinner primary melanoma (less than 1.5 mm; HR, 0.46; 95% CI, 0.27-0.78; P = .004), axillary basin (HR, 0.61; 95% CI, 0.44-0.86; P = .005), fewer positive SLNs (1 vs 3 or more; HR, 0.32; 95% CI, 0.14-0.75; P = .008), and SLN tumor burden (measured by diameter less than 1 mm [HR, 0.39; 95% CI, 0.26-0.60; P = .001] or less than 5% area [HR, 0.36; 95% CI, 0.24-0.54; P < .001]). By multivariable analysis, younger age (HR, 0.57; 95% CI, 0.39-0.84; P = .004), thinner primary melanoma (HR, 0.40; 95% CI, 0.22-0.70; P = .002), axillary basin (HR, 0.55; 95% CI, 0.31-0.96; P = .03), SLN metastasis diameter less than 1 mm (HR, 0.52; 95% CI, 0.33-0.81; P = .007), and area less than 5% (HR, 0.58; 95% CI, 0.38-0.88; P = .01) were associated with basin control. When looking at the identified risk factors of age (50 years or older), ulceration, Breslow thickness greater than 3.5 mm, nonaxillary basin, and tumor burden of maximum diameter of 1 mm or greater and/or metastasis area of 5% or greater and excluding missing value cases, basin disease-free rates at 5 years were 96% (95% CI, 88-100) for patients with 0 risk factors, 89% (95% CI, 82-96) for 1 risk factor, 86% (95% CI, 80-93) for 2 risk factors, 80% (95% CI, 71-89) for 3 risk factors, 61% (95% CI, 48-74) for 4 risk factors, and 54% (95% CI, 36-72) for 5 or 6 risk factors. This randomized clinical trial was the largest prospective evaluation of long-term regional basin control in patients with melanoma who had nodal observation after removal of a positive SLN. SLN biopsy without CLND cleared disease in the affected nodal basin in most patients, even those with multiple risk factors for in-basin recurrence. In addition to its well-validated value in staging, SLN biopsy may also be regarded as therapeutic in some patients. ClinicalTrials.gov Identifier: NCT00297895