Impact of baseline yellow plaque assessed by coronary angioscopy on vascular response after stent implantation.

BACKGROUND: The relationship between baseline yellow plaque (YP) and vascular response after stent implantation has not been fully investigated. METHODS: This was a sub-analysis of the Collaboration-1 study (multicenter, retrospective, observational study). A total of 88 lesions from 80 patients with chronic coronary syndrome who underwent percutaneous coronary intervention were analyzed. Optical coherence tomography (OCT) and coronary angioscopy (CAS) were serially performed immediately and 11 months after stent implantation. YP was defined as the stented segment with yellow or intensive yellow color assessed by CAS. Neoatherosclerosis was defined as a lipid or calcified neointima assessed by OCT. OCT and CAS findings at 11 months were compared between lesions with baseline YP (YP group) and lesions without baseline YP (Non-YP group). RESULTS: Baseline YP was detected in 37 lesions (42 %). OCT findings at 11 months showed that the incidence of neoatherosclerosis was significantly higher in the YP group (11 % versus 0 %, p = 0.028) and mean neointimal thickness tended to be lower (104 ±â€¯43 µm versus 120 ±â€¯48 µm, p = 0.098). CAS findings at 11 months demonstrated that the dominant and minimum neointimal coverage grades were significantly lower (p = 0.049 and P = 0.026) and maximum yellow color grade was significantly higher (p < 0.001) in the YP group. CONCLUSIONS: Baseline YP affected the incidence of neoatherosclerosis as well as poor neointimal coverage at 11 months after stent implantation.

Fusion of amyloid beta with ferritin yields an isolated oligomeric beta-sheet-rich aggregate inside the ferritin cage.

Alzheimer's disease is a severe brain condition caused by the formation of amyloid plaques composed of amyloid beta (Aß) peptides. These peptides form oligomers, protofibrils, and fibrils before deposition into amyloid plaques. Among these intermediates, Aß oligomers (AßOs) were found to be the most toxic and therefore an appealing target for drug development and understanding their role in the disease. However, precise isolation and characterization of AßOs have proven challenging because AßOs tend to aggregate and form heterogeneous mixtures in solution. As a solution, we genetically fused the Aß peptide with a ferritin monomer. Such fusion allowed the encapsulation of precisely 24 Aß peptides inside the 24-mer ferritin cage. Using high-speed atomic force microscopy (HS-AFM), we disassembled ferritin and directly visualized the Aß core enclosed within the cage. The thioflavin-T assay (ThT) and attenuated total reflection infrared spectroscopy (ATR-IR) revealed the presence of a ß-sheet structure in the encapsulated oligomeric aggregate. Gallic acid, an amyloid inhibitor, can inhibit the fluorescence of ThT bound AßOs. Our approach represents a significant advancement in the isolation and characterization of ß-sheet rich AßOs and is expected to be useful for future studies of other disordered peptides such as α-synuclein and tau.

Crystal structure of the in-cell Cry1Aa purified from Bacillus thuringiensis.

In-cell protein crystals which spontaneously crystallize in living cells, have recently been analyzed in investigations of their structures and biological functions. The crystals have been challenging to analyze structurally because of their small size. Therefore, the number of in-cell protein crystals in which the native structure has been determined is limited because most of the structures of in-cell crystals have been determined by recrystallization after dissolution. Some proteins have been reported to form intermolecular disulfide bonds in natural protein crystals that stabilize the crystals. Here, we focus on Cry1Aa, a cysteine-rich protein that crystallizes in Bacillus thuringiensis (Bt) and forms disulfide bonds. Previously, the full-length structure of 135 kDa Cry1Ac, which is the same size as Cry1Aa, was determined by recrystallization of dissolved protein from crystals purified from Bt cells. However, the formation of disulfide bonds has not been investigated because it was necessary to replace cysteine residues to prevent aggregation of the soluble protein. In this work, we succeeded in direct X-ray crystallographic analysis using crystals purified from Bt cells and characterized the cross-linked network of disulfide bonds within Cry1Aa crystals.

Displaying a Protein Cage on a Protein Crystal by In-Cell Crystal Engineering.

The development of solid biomaterials has rapidly progressed in recent years in applications in bionanotechnology. The immobilization of proteins, such as enzymes, within protein crystals is being used to develop solid catalysts and functionalized materials. However, an efficient method for encapsulating protein assemblies has not yet been established. This work presents a novel approach to displaying protein cages onto a crystalline protein scaffold using in-cell protein crystal engineering. The polyhedra crystal (PhC) scaffold, which displays a ferritin cage, was produced by coexpression of polyhedrin monomer (PhM) and H1-ferritin (H1-Fr) monomer in Escherichia coli. The H1-tag is derived from the H1-helix of PhM. Our technique represents a unique strategy for immobilizing protein assemblies onto in-cell protein crystals and is expected to contribute to various applications in bionanotechnology.

In-Cell Engineering of Protein Crystals into Hybrid Solid Catalysts for Artificial Photosynthesis.

The emergence of protein-based crystalline materials offers promising opportunities in enzyme immobilization. However, the current systems used for encapsulation of protein crystals are limited to either exogenous small molecules or monomeric proteins. In this work, polyhedra crystals were used to simultaneously encapsulate the foreign enzymes FDH and the organic photocatalyst eosin Y. These hybrid protein crystals are prepared easily by cocrystallization within a cell without a requirement for complex purification processes because they spontaneously form 1 µm scale solid particles. After immobilization within protein crystals, the recombinant FDH is recyclable and thermally stable and maintains 94.4% activity compared to the free enzyme. In addition, the incorporation of eosin Y endows the solid catalyst with CO2-formate conversion activity based on a cascade reaction. This work indicates that engineering protein crystals by both in vivo and in vitro strategies will provide robust and environmentally friendly solid catalysts for artificial photosynthesis.

A Generalized Method for Metal Fixation in Horse Spleen L-Ferritin Cage.

The naturally occurring iron storage protein, ferritin, has been recognized as an important template for preparing inorganic nanomaterials by fixation of metal ions and metal complexes into the cage. Such ferritin-based biomaterials find applications in various fields like bioimaging, drug delivery, catalysis, and biotechnology. The unique structural features with exceptional stability at high temperature up to ca. 100 °C and a wide pH range of 2-11 enable to design the ferritin cage for such interesting applications. Infiltration of metals into ferritin is one of the key steps for preparing ferritin-based inorganic bionanomaterials. Metal-immobilized ferritin cage can be directly utilized for applications or act as a precursor for synthesizing monodisperse and water-soluble nanoparticles. Considering this, herein, we have described a general protocol on how to immobilize metal into a ferritin cage and crystallize the metal composite for structure determination.

Cross-Linked Crystals of Dirhodium Tetraacetate/RNase A Adduct Can Be Used as Heterogeneous Catalysts.

Due to their unique coordination structure, dirhodium paddlewheel complexes are of interest in several research fields, like medicinal chemistry, catalysis, etc. Previously, these complexes were conjugated to proteins and peptides for developing artificial metalloenzymes as homogeneous catalysts. Fixation of dirhodium complexes into protein crystals is interesting to develop heterogeneous catalysts. Porous solvent channels present in protein crystals can benefit the activity by increasing the probability of substrate collisions at the catalytic Rh binding sites. Toward this goal, the present work describes the use of bovine pancreatic ribonuclease (RNase A) crystals with a pore size of 4 nm (P3221 space group) for fixing [Rh2(OAc)4] and developing a heterogeneous catalyst to perform reactions in an aqueous medium. The structure of the [Rh2(OAc)4]/RNase A adduct was investigated by X-ray crystallography: the metal complex structure remains unperturbed upon protein binding. Using a number of crystal structures, metal complex accumulation over time, within the RNase A crystals, and structures at variable temperatures were evaluated. We also report the large-scale preparation of microcrystals (∼10-20 µm) of the [Rh2(OAc)4]/RNase A adduct and cross-linking reaction with glutaraldehyde. The catalytic olefin cyclopropanation reaction and self-coupling of diazo compounds by these cross-linked [Rh2(OAc)4]/RNase A crystals were demonstrated. The results of this work reveal that these systems can be used as heterogeneous catalysts to promote reactions in aqueous solution. Overall, our findings demonstrate that the dirhodium paddlewheel complexes can be fixed in porous biomolecule crystals, like those of RNase A, to prepare biohybrid materials for catalytic applications.

Elucidating Conformational Dynamics and Thermostability of Designed Aromatic Clusters by Using Protein Cages.

Multiple aromatic residues assemble to form higher ordered structures known as "aromatic clusters" in proteins and play essential roles in biological systems. However, the stabilization mechanism and dynamic behavior of aromatic clusters remain unclear. This study describes designed aromatic interactions confined within a protein cage to reveal how aromatic clusters affect protein stability. The crystal structures and calorimetric measurements indicate that the formation of inter-subunit phenylalanine clusters enhance the interhelix interactions and increase the melting temperature. Theoretical calculations suggest that this is caused by the transformation of the T-shaped geometry into π-π stacking at high temperatures, and the hydration entropic gain. Thus, the isolated nanoenvironment in a protein cage allows reconstruction and detailed analysis of multiple clustering residues for elucidating the mechanisms of various biomolecular interactions in nature which can be applied to design of bionanomaterials.

Specific COVID-19 risk behaviors and the preventive effect of personal protective equipment among healthcare workers in Japan.

As coronavirus disease 2019 (COVID-19) outbreaks in healthcare facilities are a serious public health concern, we performed a case-control study to investigate the risk of COVID-19 infection in healthcare workers. We collected data on participants' sociodemographic characteristics, contact behaviors, installation status of personal protective equipment, and polymerase chain reaction testing results. We also collected whole blood and assessed seropositivity using the electrochemiluminescence immunoassay and microneutralization assay. In total, 161 (8.5%) of 1,899 participants were seropositive between August 3 and November 13, 2020. Physical contact (adjusted odds ratio 2.4, 95% confidence interval 1.1-5.6) and aerosol-generating procedures (1.9, 1.1-3.2) were associated with seropositivity. Using goggles (0.2, 0.1-0.5) and N95 masks (0.3, 0.1-0.8) had a preventive effect. Seroprevalence was higher in the outbreak ward (18.6%) than in the COVID-19 dedicated ward (1.4%). Results showed certain specific risk behaviors of COVID-19; proper infection prevention practices reduced these risks.

Design of a Hierarchical Assembly at a Solid-Liquid Interface Using an Asymmetric Protein Needle.

Design and control of processes for a hierarchical assembly of proteins remain challenging because it requires consideration of design principles with atomic-level accuracy. Previous studies have adopted symmetry-based strategies to minimize the complexity of protein-protein interactions and this has placed constraints on the structures of the resulting protein assemblies. In the present work, we used an anisotropic-shaped protein needle, gene product 5 (gp5) from bacteriophage T4 with a C-terminal hexahistidine-tag (His-tag) (gp5_CHis), to construct a hierarchical assembly with two distinct protein-protein interaction sites. High-speed atomic force microscopy (HS-AFM) measurements reveal that it forms unique tetrameric clusters through its N-terminal head on a mica surface. The clusters further self-assemble into a monolayer through the C-terminal His-tag. The HS-AFM images and displacement analyses show that the monolayer is a network-like structure rather than a crystalline lattice. Our results expand the toolbox for constructing hierarchical protein assemblies based on structural anisotropy.

Engineering of an in-cell protein crystal for fastening a metastable conformation of a target miniprotein.

Protein crystals can be utilized as porous scaffolds to capture exogenous molecules. Immobilization of target proteins using protein crystals is expected to facilitate X-ray structure analysis of proteins that are difficult to be crystallized. One of the advantages of scaffold-assisted structure determination is the analysis of metastable structures that are not observed in solution. However, efforts to fix target proteins within the pores of scaffold protein crystals have been limited due to the lack of strategies to control protein-protein interactions formed in the crystals. In this study, we analyze the metastable structure of the miniprotein, CLN025, which forms a ß-hairpin structure in solution, using a polyhedra crystal (PhC), an in-cell protein crystal. CLN025 is successfully fixed within the PhC scaffold by replacing the original loop region. X-ray crystal structure analysis and molecular dynamics (MD) simulation reveal that CLN025 is fixed as a helical structure in a metastable state by non-covalent interactions in the scaffold crystal. These results indicate that modulation of intermolecular interactions can trap various protein conformations in the engineered PhC and provides a new strategy for scaffold-assisted structure determination.

Risk factor structure of heart failure in patients with cancer after treatment with anticancer agents' assessment by big data from a Japanese electronic health record.

As the prognosis of cancer patients has been improved, comorbidity of heart failure (HF) in cancer survivors is a serious concern, especially in the aged population. This study aimed to examine the risk factors of HF development after treatment by anticancer agents, using a machine learning-based analysis of a massive dataset obtained from the electronic health record (EHR) in Japan. This retrospective, cohort study, using a dataset from 2008 to 2017 in the Diagnosis Procedure Combination (DPC) database in Japan, enrolled 140,327 patients. The structure of risk factors was determined using multivariable analysis and classification and regression tree (CART) algorithm for time-to-event data. The mean follow-up period was 1.55 years. The prevalence of HF after anticancer agent administration were 4.0%. HF was more prevalent in the older than the younger. As the presence of cardiovascular diseases and various risk factors predicted HF, CART analysis of the risk factors revealed that the risk factor structures complicatedly differed among different age groups. The highest risk combination was hypertension, diabetes mellitus, and atrial fibrillation in the group aged ≤ 64 years, and the presence of ischemic heart disease was a key in both groups aged 65-74 years and 75 ≤ years. The machine learning-based approach was able to develop complicated HF risk structures in cancer patients after anticancer agents in different age population, of which knowledge would be essential for realizing precision medicine to improve the prognosis of cancer patients.

Cell-free protein crystallization for nanocrystal structure determination.

In-cell protein crystallization (ICPC) has been investigated as a technique to support the advancement of structural biology because it does not require protein purification and a complicated crystallization process. However, only a few protein structures have been reported because these crystals formed incidentally in living cells and are insufficient in size and quality for structure analysis. Here, we have developed a cell-free protein crystallization (CFPC) method, which involves direct protein crystallization using cell-free protein synthesis. We have succeeded in crystallization and structure determination of nano-sized polyhedra crystal (PhC) at a high resolution of 1.80 Å. Furthermore, nanocrystals were synthesized at a reaction scale of only 20 µL using the dialysis method, enabling structural analysis at a resolution of 1.95 Å. To further demonstrate the potential of CFPC, we attempted to determine the structure of crystalline inclusion protein A (CipA), whose structure had not yet been determined. We added chemical reagents as a twinning inhibitor to the CFPC solution, which enabled us to determine the structure of CipA at 2.11 Å resolution. This technology greatly expands the high-throughput structure determination method of unstable, low-yield, fusion, and substrate-biding proteins that have been difficult to analyze with conventional methods.

Comparison of 1-Month and 12-Month Vessel Responses Between the Polymer-Free Biolimus A9-Coated Stent and the Durable Polymer Everolimus-Eluting Stent.

BACKGROUND: A polymer-free biolimus A9-coated stent (PF-BCS) may achieve better arterial healing than a durable polymer drug-eluting stent owing to its polymer-free feature.Methods and Results: This multicenter, prospective, observational study enrolled 105 patients (132 lesions) who underwent PF-BCS (51 patients, 71 lesions) or durable polymer everolimus-eluting stent (DP-EES, 54 patients, 61 lesions) implantation. Serial coronary angioscopy (CAS) and optical coherence tomography (OCT) examinations were performed at 1 and 12 months, and the serial vessel responses were compared between PF-BCS and DP-EES. The primary outcome measure was the incidence of subclinical intrastent thrombus on CAS. The secondary outcome measures were: adequate strut coverage (≥40 µm) on OCT and maximum yellow color grade on CAS. The incidence of thrombus was high at 1 month (100% vs. 93%, P=0.091), but decreased at 12 months (18% vs. 25%, P=0.56), without a significant difference between PF-BCS and DP-EES. The adequate strut coverage rate was significantly higher (84±14% vs. 69±22%, P<0.001) and yellow color was significantly less intense (P=0.012) at 12 months in PF-BCS than in DP-EES; however, they were not significantly different at 1 month (adequate strut coverage: 47±21% vs. 50±17%, P=0.40; yellow color: P=0.99). CONCLUSIONS: Although the thrombogenicity of PF-BCS was similar to that of DP-EES, the adequate coverage and plaque stabilization rates of PF-BCS were superior to those of DP-EES at 12 months.

Changes in Antithrombotic Therapy Over Time and Durability of a Prasugrel WOEST-Like Regimen for Percutaneous Coronary Intervention Patients With Atrial Fibrillation　- Post Hoc Analysis of the PENDULUM Mono and PENDULUM Registries.

Background: Previously published randomized atrial fibrillation (AF) percutaneous coronary intervention (PCI) trials have demonstrated the safety and efficacy of a WOEST-like regimen (oral anticoagulant [OAC] plus P2Y12 inhibitor) in patients with AF PCI within 1 year. However, the efficacy of this regimen in real-world practice has not been fully confirmed, especially the efficacy of the WOEST-like regimen using the approved dose of prasugrel in Japan. Methods and Results: This post hoc analysis included 186 and 220 patients from the PENDULUM mono and PENDULUM registries, respectively. Endpoints were the cumulative incidences of clinically relevant bleeding (CRB) and major adverse cardiac and cerebrovascular events (MACCE) at 12 months after PCI. Differences in the enrollment period led to an increase in OAC prescriptions (from 64.7% to 81.2%) and a reduction in the median duration of triple antithrombotic therapy (from 203.0 to 32.0 days) in the PENDULUM vs. PENDULUM mono registries, respectively. After adjustment by the inverse probability of treatment method, in patients with OAC, PENDULUM mono AF significantly reduced CRB without increasing MACCE compared with PENDULUM AF. Conclusions: A WOEST-like regimen with prasugrel may reduce CRB, without increasing MACCE, in Japanese patients with AF and high bleeding risk undergoing PCI.

Early P2Y12 Inhibitor Single Antiplatelet Therapy for High-Bleeding Risk Patients After Stenting　- PENDULUM Mono 24-Month Analysis.

BACKGROUND: In PENDULUM mono, Japanese patients with high bleeding risk (HBR) received short-term dual antiplatelet therapy (DAPT) followed by single antiplatelet therapy (SAPT) with prasugrel after percutaneous coronary intervention (PCI). One-year data from PENDULUM mono showed better outcomes with prasugrel monotherapy after short-term DAPT compared with matched patients in the PENDULUM registry with longer DAPT durations according to guidelines at that time. This study presents 2-year results.Methods and Results: We compared 24-month data from PENDULUM mono (n=1,107; de-escalation strategy group) and the PENDULUM registry (n=2,273; conventional strategy group); both were multicenter, non-interventional, prospective registry studies, using the inverse probability of treatment weighting (IPTW) method. In the PENDULUM mono group, the cumulative incidence of clinically relevant bleeding (CRB) at 24 months post-PCI (primary endpoint) was 6.8%, and that of major adverse cardiac and cerebrovascular events (MACCE) was 8.9%. After IPTW adjustment, the cumulative incidence of CRB was 5.8% and 7.2% in PENDULUM mono and the PENDULUM registry, respectively (hazard ratio [HR] 0.77; 95% confidence interval [CI] 0.57-1.04; P=0.086), and that of MACCE was 8.0% and 9.5%, respectively (HR 0.77; 95% CI 0.59-1.01; P=0.061). CONCLUSIONS: Japanese PCI patients with HBR prescribed prasugrel SAPT after short-term DAPT had a lower ischemic event risk than those prescribed long-term DAPT, and this was particularly relevant for ischemic events after 1 year.

Clinical Outcomes After Percutaneous Coronary Intervention in East Asian Patients　- 30-Month Results of the PENDULUM Registry.

BACKGROUND: The 12-month results of the PENDULUM registry showed that after implantation of second-generation drug-eluting stents (DES), high P2Y12reaction unit (HPR) were independently associated with ischemic but not bleeding events.Methods and Results: This study analyzed cumulative incidences of major adverse cardiac and cerebrovascular events (MACCE) and major bleeding (Bleeding Academic Research Consortium type 3 and 5) at 30 months after index percutaneous coronary intervention (PCI) (primary endpoints). Of 6,422 patients undergoing PCI with DES, 5,796 completed the 30-month follow up. The continuation rate of dual antiplatelet therapy decreased to 59.3% at 12 months and 26.4% at 30 months. At 30 months, the cumulative incidence of MACCE increased linearly and reached 9.5% (95% confidence interval 8.8-10.2) and that of major bleeding had the inflection point at 12 months and was 4.4% (3.9-5.0). MACCE and bleeding events were higher in HPR patients (unadjusted P value). After covariate adjustment, P2Y12reactivity units measured immediately after index PCI was not an independent risk factor for MACCE or major bleeding at 30 months. CONCLUSIONS: MACCE consistently increased after 12 months post-PCI, whereas the increase in major bleeding events slowed down after 12 months in Japanese PCI patients in a real-world clinical setting. HPR patients had increased MACCE and bleeding complications, but HPR was not an independent risk factor of events at 30 months.