In situ crystal data-collection and ligand-screening system at SPring-8.

In situ diffraction data collection using crystallization plates has been utilized for macromolecules to evaluate crystal quality without requiring additional sample treatment such as cryocooling. Although it is difficult to collect complete data sets using this technique due to the mechanical limitation of crystal rotation, recent advances in methods for data collection from multiple crystals have overcome this issue. At SPring-8, an in situ diffraction measurement system was constructed consisting of a goniometer for a plate, an articulated robot and plate storage. Using this system, complete data sets were obtained utilizing the small-wedge measurement method. Combining this system with an acoustic liquid handler to prepare protein-ligand complex crystals by applying fragment compounds to trypsin crystals for in situ soaking, binding was confirmed for seven out of eight compounds. These results show that the system functioned properly to collect complete data for structural analysis and to expand the capability for ligand screening in combination with a liquid dispenser.

Trans-cuff leakage in bio-Bentall operation.

We have reported a case of trans-cuff leakage that occurred in a composite graft of bio-Bentall operation. The leakage resolved several months after surgery, similar to the trans-cuff leakage seen in simple aortic valve replacement. We have proposed hypotheses on the mechanism of trans-cuff leakage during a bio-Bentall operation and suggested ways to prevent it from occurring.

Short-lived intermediate in N2O generation by P450 NO reductase captured by time-resolved IR spectroscopy and XFEL crystallography.

Nitric oxide (NO) reductase from the fungus Fusarium oxysporum is a P450-type enzyme (P450nor) that catalyzes the reduction of NO to nitrous oxide (N2O) in the global nitrogen cycle. In this enzymatic reaction, the heme-bound NO is activated by the direct hydride transfer from NADH to generate a short-lived intermediate ( I ), a key state to promote N-N bond formation and N-O bond cleavage. This study applied time-resolved (TR) techniques in conjunction with photolabile-caged NO to gain direct experimental results for the characterization of the coordination and electronic structures of I TR freeze-trap crystallography using an X-ray free electron laser (XFEL) reveals highly bent Fe-NO coordination in I , with an elongated Fe-NO bond length (Fe-NO = 1.91 Å, Fe-N-O = 138°) in the absence of NAD+ TR-infrared (IR) spectroscopy detects the formation of I with an N-O stretching frequency of 1,290 cm-1 upon hydride transfer from NADH to the Fe3+-NO enzyme via the dissociation of NAD+ from a transient state, with an N-O stretching of 1,330 cm-1 and a lifetime of ca. 16 ms. Quantum mechanics/molecular mechanics calculations, based on these crystallographic and IR spectroscopic results, demonstrate that the electronic structure of I is characterized by a singly protonated Fe3+-NHO•- radical. The current findings provide conclusive evidence for the N2O generation mechanism via a radical-radical coupling of the heme nitroxyl complex with the second NO molecule.

XFEL Crystal Structures of Peroxidase Compound II.

Oxygen activation in all heme enzymes requires the formation of high oxidation states of iron, usually referred to as ferryl heme. There are two known intermediates: Compound I and Compound II. The nature of the ferryl heme-and whether it is an FeIV =O or FeIV -OH species-is important for controlling reactivity across groups of heme enzymes. The most recent evidence for Compound I indicates that the ferryl heme is an unprotonated FeIV =O species. For Compound II, the nature of the ferryl heme is not unambiguously established. Here, we report 1.06 Šand 1.50 Šcrystal structures for Compound II intermediates in cytochrome c peroxidase (CcP) and ascorbate peroxidase (APX), collected using the X-ray free electron laser at SACLA. The structures reveal differences between the two peroxidases. The iron-oxygen bond length in CcP (1.76 Å) is notably shorter than in APX (1.87 Å). The results indicate that the ferryl species is finely tuned across Compound I and Compound II species in closely related peroxidase enzymes. We propose that this fine-tuning is linked to the functional need for proton delivery to the heme.

XFEL Crystal Structures of Peroxidase Compound II.

Oxygen activation in all heme enzymes requires the formation of high oxidation states of iron, usually referred to as ferryl heme. There are two known intermediates: Compound I and Compound II. The nature of the ferryl heme-and whether it is an FeIV=O or FeIV-OH species-is important for controlling reactivity across groups of heme enzymes. The most recent evidence for Compound I indicates that the ferryl heme is an unprotonated FeIV=O species. For Compound II, the nature of the ferryl heme is not unambiguously established. Here, we report 1.06 Šand 1.50 Šcrystal structures for Compound II intermediates in cytochrome c peroxidase (CcP) and ascorbate peroxidase (APX), collected using the X-ray free electron laser at SACLA. The structures reveal differences between the two peroxidases. The iron-oxygen bond length in CcP (1.76 Å) is notably shorter than in APX (1.87 Å). The results indicate that the ferryl species is finely tuned across Compound I and Compound II species in closely related peroxidase enzymes. We propose that this fine-tuning is linked to the functional need for proton delivery to the heme.

Paradoxical doppler echocardiographic parameters during recovery of left atrial contractility after spontaneous conversion from paroxysmal atrial fibrillation to sinus rhythm.

Paroxysmal atrial fibrillation (PAF) reduces atrial contractility due to atrial remodeling, but little is known about the process by which contractile function is reconstituted after spontaneous conversion to sinus rhythm (SR). A 63-year-old healthy man developed PAF. PAF persisted for 2 days before spontaneous conversion to SR. Serial echocardiograms were performed at 1, 24 h, 3/4/7 days after conversion. Longitudinal myocardial strain during the pump phase of the left atrium (LA) was generally reduced at 1 h. Normal strain of the LA was restored at 3 days with the exception of the lateral wall, where restoration was delayed until 4 days. The ratio between the mitral early and atrial diastolic velocities (E/A) at 24 h was within a pseudonormal range at 1.8, but the ratio between E and early mitral annulus velocity (e': E/e') remained normal. The E/A ratio gradually decreased until 7 days post conversion, but the E/e' ratio remained normal throughout the observation period.

A rare form of cardiac myxoma: interatrial septum tumor.

A myxoma is one of the most common tumors in cardiac surgery, with most of them originating from the left or right atrial walls and causing embolic complications. An 84-year-old woman was referred to our institution after echocardiography showed an interatrial septum tumor that was presumed to be a thrombus; however, postoperative pathological examination revealed it to be a cardiac myxoma. This atypical myxoma was covered with a solid capsule containing connective tissue and substantial calcifications, which had made it difficult to establish a diagnosis of myxoma.

Development of SPACE-II for rapid sample exchange at SPring-8 macromolecular crystallography beamlines.

Reducing the sample-exchange time is a crucial issue in maximizing the throughput of macromolecular crystallography (MX) beamlines because the diffraction data collection itself is completed within a minute in the era of pixel-array detectors. To this end, an upgraded sample changer, SPACE-II, has been developed on the basis of the previous model, SPACE (SPring-8 Precise Automatic Cryo-sample Exchanger), at the BL41XU beamline at SPring-8. SPACE-II achieves one sample-exchange step within 16 s, of which its action accounts for only 11 s, because of three features: (i) the implementation of twin arms that enable samples to be exchanged in one cycle of mount-arm action, (ii) the implementation of long-stroke mount arms that allow samples to be exchanged without withdrawal of the detector and (iii) the use of a fast-moving translation and rotation stage for the mount arms. By pre-holding the next sample prior to the sample-exchange sequence, the time was further decreased to 11 s in the case of automatic data collection, of which the action of SPACE-II accounted for 8 s. Moreover, the sample capacity was expanded from four to eight Uni-Pucks. The performance of SPACE-II has been demonstrated in over two years of operation at BL41XU; the average number of samples mounted on the diffractometer in one day was increased from 132 to 185, with an error rate of 0.089%, which counted incidents in which users could not continue with an experiment without recovery work by entering the experimental hutch. On the basis of these results, SPACE-II has been installed at three other MX beamlines at SPring-8 as of July 2019. The fast and highly reliable SPACE-II is now one of the most important pieces of infrastructure for the MX beamlines at SPring-8, providing users with the opportunity to fully make use of limited beamtime with brilliant X-rays.

An oxyl/oxo mechanism for oxygen-oxygen coupling in PSII revealed by an x-ray free-electron laser.

Photosynthetic water oxidation is catalyzed by the Mn4CaO5 cluster of photosystem II (PSII) with linear progression through five S-state intermediates (S0 to S4). To reveal the mechanism of water oxidation, we analyzed structures of PSII in the S1, S2, and S3 states by x-ray free-electron laser serial crystallography. No insertion of water was found in S2, but flipping of D1 Glu189 upon transition to S3 leads to the opening of a water channel and provides a space for incorporation of an additional oxygen ligand, resulting in an open cubane Mn4CaO6 cluster with an oxyl/oxo bridge. Structural changes of PSII between the different S states reveal cooperative action of substrate water access, proton release, and dioxygen formation in photosynthetic water oxidation.

Eicosapentaenoic acid therapy is associated with decreased coronary plaque instability assessed using optical frequency domain imaging.

BACKGROUND: The relationship between eicosapentaenoic acid (EPA) therapy and coronary plaque stability assessed by optical frequency domain imaging (OFDI) has not been thoroughly described. HYPOTHESIS: EPA therapy is associated with decreased plaque instability in patients undergoing percutaneous coronary intervention (PCI) using OFDI. METHODS: Data on coronary artery plaques from 121 patients who consecutively underwent PCI between October 2015 and July 2018 were retrospectively analyzed. Of these patients, 109 were untreated (no-EPA group), whereas 12 were treated with EPA (EPA group). Each plaque's morphological characteristics were analyzed using OFDI. RESULTS: We used 1:4 propensity score matching for patients who received or did not receive EPA therapy before PCI. Baseline characteristics were balanced between both groups (age, sex, body mass index, diabetes mellitus, hypertension, dyslipidemia, chronic kidney disease, smoking, previous PCI or coronary artery bypass grafting, previous myocardial infarction, prior statin use, acute coronary syndrome, hemoglobin A1c level, low-density lipoprotein cholesterol concentration, triglyceride concentration, and high-density lipoprotein cholesterol concentration). OFDI data from 60 patients were analyzed in this study. The EPA group had significantly lower mean lipid index (818 ± 806 vs 1574 ± 891) and macrophage grade (13.5 ± 5.9 vs 19.3 ± 7.4) but higher mean minimum fibrous cap thickness (109.2 ± 55.7 vs 81.6 ± 36.4 µm) than the no-EPA group (P = 0.010, 0.019, and 0.040, respectively). Multiple logistic regression analyses showed that prior EPA use was independently associated with lower lipid index and macrophage grade (P = 0.043 and 0.024, respectively). CONCLUSION: This OFDI analysis suggests that EPA therapy is associated with decreased plaque instability in patients undergoing PCI.

[Surgical Repair of Stanford Type A Acute Aortic Dissection Associated with Anomalous Retroaortic Left Brachiocephalic Vein;Report of a Case].

Anomalous retroaortic left brachiocephalic vein( ARLBV) is a rare vascular abnormaly, usually asossiated with congenital heart diseases. We present a very rare case of Stanford type A acute aortic dissection associated with ARLBV. A 70-year-old man was referred to our hospital complaining of worsening back pain and chest pain. Computed tomographic angiogram showed Stanford type A acute aortic dissection from the ascending aorta to the distal abdominal aorta. Furthermore, systemic venous anomalies were revealed. The left brachiocephalic vein coursed the left side of the aortic arch, anterior to the central pulmonary artery, and posterior to the ascending aorta, and joined the right brachiocephalic vein. Replacement of the ascending aorta was performed in an emergency, and the postoperative course was uneventful.

Obstructive sleep apnea is associated with increased coronary plaque instability: an optical frequency domain imaging study.

Obstructive sleep apnea (OSA) is associated with coronary artery disease (CAD) and with an increased risk for myocardial infarction, stroke or death due to cardiovascular disease. Optical frequency-domain imaging (OFDI) is a useful modality for evaluating the characteristics of atherosclerotic plaque. The purpose of the study was to use OFDI to investigate the association of OSA with coronary plaque characteristics in patients undergoing percutaneous coronary intervention (PCI). We retrospectively analyzed OFDI data for coronary artery plaques from 15 patients with OSA and 35 non-OSA patients treated between October 2015 and October 2018. Plaque morphology was evaluated for 70 lesions, including 21 from patients with OSA and 49 from non-OSA patients. Compared with the non-OSA group, patients with OSA had significantly higher prevalences of thinned cap fibroatheroma (TCFA) (67% vs. 35%, P = 0.014) and microchannels (86% vs. 55%, P = 0.014); a significantly higher mean lipid index (1392 ± 982 vs. 817 ± 699, P = 0.021), macrophage grade (8.4 ± 6.4 vs. 4.8 ± 4.5, P = 0.030), and maximum number of microchannels (1.5 ± 1.0 vs. 0.7 ± 0.7, P = 0.001); and a significantly lower mean minimum fibrous cap thickness (69.4 ± 28.7 vs. 96.1 ± 51.8 µm, P = 0.008). This OFDI analysis suggests that OSA is associated with unstable plaque characteristics in patients with CAD. More intensive medical management for stabilization of coronary atherosclerotic plaque is required in patients with OSA.

Woman in her 50s with shortness of breath on exertion.

CLINICAL INTRODUCTION: A 59-year-old woman visited an outpatient cardiology clinic due to shortness of breath on exertion. Physical examination showed no significant abnormality of vital signs. A III/VI systolic murmur was heard on the fourth intercostal space at the right sternal border. The majority of laboratory tests were normal. Chest X-ray showed a curved vessel shadow (figure 1A). Initial transthoracic echocardiography showed abnormal blood flow into the inferior vena cava (IVC) in the subxiphoid long axis view (figure 1B) and mild right heart dilatation (online supplementary figure 1). Transoesophageal echocardiography showed severe tricuspid regurgitation (online supplementary figure 2).heartjnl;105/2/110/F1F1F1Figure 1(A) Chest X-ray. (B) Colour Doppler image in the subxiphoid long axis view.DC1SP110.1136/heartjnl-2018-313655.supp1Supplementary data DC2SP210.1136/heartjnl-2018-313655.supp2Supplementary data QUESTION: What is the most likely underlying disease for the patient's shortness of breath on exertion?Pulmonary arteriovenous fistula.Pulmonary arterial hypertension.Lung cancer.Partial anomalous pulmonary venous connection.Isolated tricuspid regurgitation.

[Video-assisted Thoracoscopic Pericardial Fenestration for Pericardial Effusion in a Patient with Crow-Fukase Syndrome].

A 65-year-old woman was referred to our department with recurrent pericardial effusion. Her serum vascular endothelial growth factor was high, serum M-protein was positive, and nerve conduction velocity of extremities was decreased. Therefore, she was diagnosed with Crow-Fukase (polyneuropathy, organomegaly, endocrinopathy, M protein, skin changes:POEMS) syndrome, which is characterized by the presence of plasma cell dyscrasia with them. We performed video-thoracoscopic pericardial fenestration with 4×4 cm window. The postoperative course was uneventful, and the pericardial effusion completely disappeared. Video-assisted thoracoscopic pericardial fenestration was a safe and effective treatment for recurrent pericardial effusion.

Investigation on the optimal implantation site and setting of Reveal LINQ® avoiding interference with performance of transthoracic echocardiography.

BACKGROUND: The optimal implantation site of a new implantable cardiac monitor (ICM) named Reveal LINQ® may be limited based on a sufficient amplitude of R wave potential (AEP) acquisition because it is the same anatomic area used for transthoracic echocardiography (TTE). METHODS: Among 18 healthy volunteers, we assessed AEPs in 3 combinations through parasternal placement of 2 electrodes, (i) in the 4th intercostal space (ICS; site A/setting a; A/a), (ii) the same setting in the 5th ICS (site B/setting a; B/a), and (iii) in a sagittal plane relative to the left sternal border at the 4th ICS (site A/setting b; A/b), and further measured AFPs in several body positions in all site-setting combinations: supine, left and right lateral decubitus, sitting, and standing. The degree of interference with TTE performance was assessed by placement of an imitation ICM in setting a at both sites A and B. RESULTS: Only the AEPs in A/a and B/a met the criteria (AEP ≥ 0.3 mV) in all positions. The AEPs in the supine position with all combinations were higher than those achieved in other positions (P < .001). The imitation interfered with TTE performance at site A among 78% of subjects, but only 17% at site B (P = .0006). The end-diastolic dimension of the left ventricle at site A was decreased after the imitation placement (P = .028). At site B, all female subjects complained of discomfort because their brassieres overlaid the imitation. CONCLUSION: The B/a combination is optimal; however, the personal discomfort related to brassieres should be considered.

Capturing an initial intermediate during the P450nor enzymatic reaction using time-resolved XFEL crystallography and caged-substrate.

Time-resolved serial femtosecond crystallography using an X-ray free electron laser (XFEL) in conjunction with a photosensitive caged-compound offers a crystallographic method to track enzymatic reactions. Here we demonstrate the application of this method using fungal NO reductase, a heme-containing enzyme, at room temperature. Twenty milliseconds after caged-NO photolysis, we identify a NO-bound form of the enzyme, which is an initial intermediate with a slightly bent Fe-N-O coordination geometry at a resolution of 2.1 Å. The NO geometry is compatible with those analyzed by XFEL-based cryo-crystallography and QM/MM calculations, indicating that we obtain an intact Fe3+-NO coordination structure that is free of X-ray radiation damage. The slightly bent NO geometry is appropriate to prevent immediate NO dissociation and thus accept H- from NADH. The combination of using XFEL and a caged-compound is a powerful tool for determining functional enzyme structures during catalytic reactions at the atomic level.

A nanosecond time-resolved XFEL analysis of structural changes associated with CO release from cytochrome c oxidase.

Bovine cytochrome c oxidase (CcO), a 420-kDa membrane protein, pumps protons using electrostatic repulsion between protons transferred through a water channel and net positive charges created by oxidation of heme a (Fe a ) for reduction of O2 at heme a3 (Fe a3). For this process to function properly, timing is essential: The channel must be closed after collection of the protons to be pumped and before Fe a oxidation. If the channel were to remain open, spontaneous backflow of the collected protons would occur. For elucidation of the channel closure mechanism, the opening of the channel, which occurs upon release of CO from CcO, is investigated by newly developed time-resolved x-ray free-electron laser and infrared techniques with nanosecond time resolution. The opening process indicates that CuB senses completion of proton collection and binds O2 before binding to Fe a3 to close the water channel using a conformational relay system, which includes CuB, heme a3, and a transmembrane helix, to block backflow of the collected protons.

Usefulness of the echocardiographic paravertebral approach for the diagnosis of descending thoracic aortic dissection.

BACKGROUND: Transthoracic echocardiography (TTE) is not recommended as the first-line diagnostic modality for Stanford type B aortic dissection (type-B AD). PURPOSES: The aims of this study were to evaluate the usefulness and factors influencing for the diagnosis of type-B AD using the transthoracic echocardiographic paravertebral approach (PVA). METHODS: We compared the image acquisition rate of descending thoracic aorta (DTA) and the diagnostic rate of type-B AD using TTE versus PVA. Both tests were compared with type-B AD, which was diagnosed by enhanced computed tomography (CT), as the reference standard. We also analyzed the factors influencing adequate image acquisition and the diagnosis of type-B AD using the PVA. The length between the dorsal thoracic surface and the DTA (TDAL) and thickness of lung on the TDAL line (LTh) were measured on the CT images. RESULTS: No significant difference was found between the image acquisition rate of the DTA between the PVA and the TTE (70.1 vs. 64.2%, p = 0.56), while the diagnostic rate of type-B AD using the PVA was significantly greater than when using the TTE (56.7 vs. 26.9%, p < 0.001). Furthermore, when adequate images of DTA were obtained using the PVA, 80.9% of the patients were diagnosed with type-B AD. A multivariate analysis demonstrated that a lower LTh (p = 0.001) and the existence of a pleural effusion (p = 0.03) significantly influenced the diagnosis of type-B AD using the PVA. CONCLUSIONS: The PVA might be a useful method for diagnosis of type-B AD, when adequate images of the DTA are obtained.

A nearly on-axis spectroscopic system for simultaneously measuring UV-visible absorption and X-ray diffraction in the SPring-8 structural genomics beamline.

UV-visible absorption spectroscopy is useful for probing the electronic and structural changes of protein active sites, and thus the on-line combination of X-ray diffraction and spectroscopic analysis is increasingly being applied. Herein, a novel absorption spectrometer was developed at SPring-8 BL26B2 with a nearly on-axis geometry between the X-ray and optical axes. A small prism mirror was placed near the X-ray beamstop to pass the light only 2° off the X-ray beam, enabling spectroscopic analysis of the X-ray-exposed volume of a crystal during X-ray diffraction data collection. The spectrometer was applied to NO reductase, a heme enzyme that catalyzes NO reduction to N2O. Radiation damage to the heme was monitored in real time during X-ray irradiation by evaluating the absorption spectral changes. Moreover, NO binding to the heme was probed via caged NO photolysis with UV light, demonstrating the extended capability of the spectrometer for intermediate analysis.