Oxygen-evolving photosystem II structures during S1-S2-S3 transitions.

Photosystem II (PSII) catalyses the oxidation of water through a four-step cycle of Si states (i = 0-4) at the Mn4CaO5 cluster1-3, during which an extra oxygen (O6) is incorporated at the S3 state to form a possible dioxygen4-7. Structural changes of the metal cluster and its environment during the S-state transitions have been studied on the microsecond timescale. Here we use pump-probe serial femtosecond crystallography to reveal the structural dynamics of PSII from nanoseconds to milliseconds after illumination with one flash (1F) or two flashes (2F). YZ, a tyrosine residue that connects the reaction centre P680 and the Mn4CaO5 cluster, showed structural changes on a nanosecond timescale, as did its surrounding amino acid residues and water molecules, reflecting the fast transfer of electrons and protons after flash illumination. Notably, one water molecule emerged in the vicinity of Glu189 of the D1 subunit of PSII (D1-E189), and was bound to the Ca2+ ion on a sub-microsecond timescale after 2F illumination. This water molecule disappeared later with the concomitant increase of O6, suggesting that it is the origin of O6. We also observed concerted movements of water molecules in the O1, O4 and Cl-1 channels and their surrounding amino acid residues to complete the sequence of electron transfer, proton release and substrate water delivery. These results provide crucial insights into the structural dynamics of PSII during S-state transitions as well as O-O bond formation.

Comparison of the rate of concomitant proximal venous stenosis between the upper and lower extremities in patients with secondary lymphedema undergoing lymphaticovenous anastomosis.

BACKGROUND: Concomitant iatrogenic proximal venous stenosis increases venous pressure and can be a risk factor for unfavorable outcomes of lymphaticovenular anastomosis (LVA) in extremities with secondary lymphedema. This study investigated the frequency and relevant factors of venous stenosis in patients diagnosed with secondary lymphedema who underwent LVA. METHODS: Patients who underwent preoperative computed tomographic venography (CTV) and LVA for secondary lymphedema of the extremities from October 2018 to March 2022 were included. The incidence of proximal venous stenosis in the affected limb on preoperative CTV and the rate of endovascular intervention were compared between upper and lower extremities. Factors affecting proximal venous stenosis were identified through multivariable analysis using independent variables, including patient age, body mass index, comorbidities, smoking history, radiation therapy, duration of lymphedema, and location of lymphedema. RESULTS: A total of 211 patients were analyzed, including 83 patients with upper extremity and 128 patients with lower extremity lymphedema. The incidence of proximal venous stenosis in the preoperative CTV was 32.5% and 7.8% in upper extremity, and lower extremity lymphedema, respectively (P < .001). The incidence of venous stenosis requiring endovascular intervention was significantly higher in the upper extremity compared with the lower extremity (16.9% vs 6.3%; P = .014). In multivariable analysis, risk factors affecting incidence of venous stenosis requiring endovascular intervention was the patient age (P = .007) and upper extremity (P = .009). CONCLUSIONS: Preoperative evaluation and treatment of venous stenosis in extremities with secondary lymphedema are necessary before LVA surgery, particularly in upper extremity lymphedema.

Perfusion area versus volume of the DIEP flap: A multivariable analysis of perforator and flap characteristics for estimation of perfusion area and volume.

BACKGROUND: The extent of perfusion of a deep inferior epigastric artery perforator (DIEP) flap is a primary concern for surgeons. This study aimed to determine whether the flap area or volume can be estimated using perforator and flap characteristics. METHODS: Intraoperative flap perfusion was assessed using indocyanine green angiography in patients who underwent DIEP flap breast reconstruction between November 2018 and February 2023. The area perfused by a single dominant perforator was delineated on the surface of the flap and measured using the ImageJ software. Multiple linear regression analysis was conducted to estimate the 'perfusion ratio,' defined as the perfused area divided by the total flap area. Potential predictor variables included flap size (cm2), flap thickness (mm), perforator diameter (mm), perforator rows (medial/lateral), vertical location of perforator (at or above/below the umbilicus), and perforator eccentricity (vertical distance from upper flap margin to perforator, cm). RESULTS: In total, 101 patients were included in this analysis. The mean 'perfusion ratio' was 67.8% ± 11.5%, predicted by perforator diameter (p = 0.022) and vertical location below umbilicus (p < 0.001) with positive correlations and negatively correlated with flap thickness (p = 0.003) in the multivariable analysis. Both perfusion area and weight were predicted by perforator diameter, vertical location of perforator, flap size, and flap thickness (p < 0.001). The coefficient of determination (adjusted R2) for prediction of perfusion weight was higher than that for the perfusion area (75.5% vs. 69.4%). CONCLUSIONS: Flap volume, rather than area, is determined by a perforator of a given diameter and location.

Real-time observation of a metal complex-driven reaction intermediate using a porous protein crystal and serial femtosecond crystallography.

Determining short-lived intermediate structures in chemical reactions is challenging. Although ultrafast spectroscopic methods can detect the formation of transient intermediates, real-space structures cannot be determined directly from such studies. Time-resolved serial femtosecond crystallography (TR-SFX) has recently proven to be a powerful method for capturing molecular changes in proteins on femtosecond timescales. However, the methodology has been mostly applied to natural proteins/enzymes and limited to reactions promoted by synthetic molecules due to structure determination challenges. This work demonstrates the applicability of TR-SFX for investigations of chemical reaction mechanisms of synthetic metal complexes. We fix a light-induced CO-releasing Mn(CO)3 reaction center in porous hen egg white lysozyme (HEWL) microcrystals. By controlling light exposure and time, we capture the real-time formation of Mn-carbonyl intermediates during the CO release reaction. The asymmetric protein environment is found to influence the order of CO release. The experimentally-observed reaction path agrees with quantum mechanical calculations. Therefore, our demonstration offers a new approach to visualize atomic-level reactions of small molecules using TR-SFX with real-space structure determination. This advance holds the potential to facilitate design of artificial metalloenzymes with precise mechanisms, empowering design, control and development of innovative reactions.