High-throughput structure determination of an intrinsically disordered protein using cell-free protein crystallization.

Intrinsically disordered proteins (IDPs) play a crucial role in various biological phenomena, dynamically changing their conformations in response to external environmental cues. To gain a deeper understanding of these proteins, it is essential to identify the determinants that fix their structures at the atomic level. Here, we developed a pipeline for rapid crystal structure analysis of IDP using a cell-free protein crystallization (CFPC) method. Through this approach, we successfully demonstrated the determination of the structure of an IDP to uncover the key determinants that stabilize its conformation. Specifically, we focused on the 11-residue fragment of c-Myc, which forms an α-helix through dimerization with a binding partner protein. This fragment was strategically recombined with an in-cell crystallizing protein and was expressed in a cell-free system. The resulting crystal structures of the c-Myc fragment were successfully determined at a resolution of 1.92 Å and we confirmed that they are identical to the structures of the complex with the native binding partner protein. This indicates that the environment of the scaffold crystal can fix the structure of c-Myc. Significantly, these crystals were obtained directly from a small reaction mixture (30 µL) incubated for only 72 h. Analysis of eight crystal structures derived from 22 mutants revealed two hydrophobic residues as the key determinants responsible for stabilizing the α-helical structure. These findings underscore the power of our CFPC screening method as a valuable tool for determining the structures of challenging target proteins and elucidating the essential molecular interactions that govern their stability.

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.

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.

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.

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.

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.

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.

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.

Protein Needles Designed to Self-Assemble through Needle Tip Engineering.

The dynamic process of formation of protein assemblies is essential to form highly ordered structures in biological systems. Advances in structural and synthetic biology have led to the construction of artificial protein assemblies. However, development of design strategies exploiting the anisotropic shape of building blocks of protein assemblies has not yet been achieved. Here, the 2D assembly pattern of protein needles (PNs) is controlled by regulating their tip-to-tip interactions. The PN is an anisotropic needle-shaped protein composed of ß-helix, foldon, and His-tag. Three different types of tip-modified PNs are designed by deleting the His-tag and foldon to change the protein-protein interactions. Observing their assembly by high-speed atomic force microscopy (HS-AFM) reveals that PN, His-tag deleted PN, and His-tag and foldon deleted PN form triangular lattices, the monomeric state with nematic order, and fiber assemblies, respectively, on a mica surface. Their assembly dynamics are observed by HS-AFM and analyzed by the theoretical models. Monte Carlo (MC) simulations indicate that the mica-PN interactions and the flexible and multipoint His-tag interactions cooperatively guide the formation of the triangular lattice. This work is expected to provide a new strategy for constructing supramolecular protein architectures by controlling directional interactions of anisotropic shaped proteins.

Importance of the Subunit-Subunit Interface in Ferritin Disassembly: A Molecular Dynamics Study.

Ferritin is a spherical cage-like protein that is useful for loading large functional particles for various applications. To our knowledge, how pH affects the interfaces inside ferritin and the mechanism of ferritin disassembly is far from complete. For this article, we conducted a series of molecular dynamics simulations (MD) at different pH values to study how interfaces affect ferritins' stability. It is shown that dimers are stable even at extremely low pH (pH 2.0), indicating that the dimer is the essential subunit for disassembly, and the slight swelling of the dimer resulting from monomer rotation inside a dimer is what triggers disassembly. During ferritin disassembly, there are two types of interfaces involved, and the interface between dimers is crucial. We also found that the driving forces for maintaining dimer stability are different when a dimer is inside ferritin and in an acidic solution. At low pH, the protonation of residues can lead to the loss of the salt bridge and the hydrogen bond between dimers, resulting in the disassembly of ferritin in an acidic environment. The above simulations reveal the possible mechanism of ferritin disassembly in an acidic solution, which can help us to design innovative and functional ferritin cages for different applications.

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.

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.

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.

Ischemic and Bleeding Events in PENDULUM Patients With High Bleeding Risk and High Platelet Reactivity.

BACKGROUND: The balance between thrombotic and bleeding risk is of great concern in high bleeding risk (HBR) patients. This study evaluated the relationship between perioperative antiplatelet reactivity and thrombotic and bleeding events in patients at HBR undergoing percutaneous coronary intervention (PCI).Methods and Results: In this post hoc analysis of the PENDULUM (Platelet rEactivity in patieNts with DrUg eLUting stent and balancing risk of bleeding and ischeMic event) registry, patients undergoing PCI were categorized as HBR or non-HBR, and stratified as having high platelet reactivity (HPR; P2Y12reaction unit [PRU] >208) or non-HPR (PRU ≤208). Cumulative incidences of cardiovascular and cerebrovascular events (Journal of the American College of Cardiology expert definitions) and bleeding events (Bleeding Academic Research Consortium criteria) were assessed 12 months after index PCI. The incidence of ischemic and bleeding events was ~3-fold higher in HBR vs. non-HBR patients. Thrombotic/ischemic events were significantly more common in the HPR subgroup in HBR patients (hazard ratio [HR]: 1.59; 95% confidence interval [CI]: 1.11-2.28; P=0.012), but there was no difference in non-HBR patients. After adjustment for covariates, HPR in HBR patients remained an independent factor for thrombotic and ischemic events (HR: 1.69; 95% CI: 1.13-2.54; P=0.011), but not for bleeding events (HR: 1.56; 95% CI: 0.78-3.11; P=0.210). CONCLUSIONS: Maintaining adequate PRU levels during PCI is an important factor in improving clinical outcomes, especially for HBR patients.

Relationship between platelet aggregation and stroke risk after percutaneous coronary intervention: a PENDULUM analysis.

In patients undergoing percutaneous coronary intervention (PCI) with a stent, high on-treatment platelet reactivity may be associated with an increased risk of stroke. This post hoc analysis of the PENDULUM registry compared the risk of post-PCI stroke according to on-treatment P2Y12 reaction unit (PRU) values. Patients aged ≥ 20 years who underwent PCI were stratified by baseline PRU (at 12 and 48 h post-PCI) as either high (HPR, > 208), optimal (OPR, > 85 to ≤ 208), or low on-treatment platelet reactivity (LPR, ≤ 85). The incidences of non-fatal ischemic and non-ischemic stroke through to 12 months post-PCI were recorded. Almost all enrolled patients (6102/6267 [97.4%]) had a risk factor for ischemic stroke, and most were receiving dual antiplatelet therapy. Of the 5906 patients with PRU data (HPR, n = 2227; OPR, n = 3002; LPR, n = 677), 47 had a non-fatal stroke post-PCI (cumulative incidence: 0.68%, ischemic; 0.18%, non-ischemic stroke). Patients with a non-fatal ischemic stroke event had statistically significantly higher post-PCI PRU values versus those without an event (P = 0.037). The incidence of non-fatal non-ischemic stroke was not related to PRU value. When the patients were stratified by PRU ≤ 153 versus > 153 at 12-48 h post-PCI, a significant difference was observed in the cumulative incidence of non-fatal stroke at 12 months (P = 0.044). We found that patients with ischemic stroke tended to have higher PRU values at 12-48 h after PCI versus those without ischemic stroke.Clinical trial registration: UMIN000020332.

Controlled Uptake of an Iridium Complex inside Engineered apo-Ferritin Nanocages: Study of Structure and Catalysis.

The effect of the mutation at the core of the ferritin nanocage (apo-rHLFr) on the uptake of IrCp* has been investigated by structural and spectroscopic methods. Site-specific mutations of two polar residues viz., Asp38 and Arg52 were investigated. The uptake of IrCp* was increased by about 1.5-fold on mutation of Arg52 by His compared to the wild-type variant, while mutation of Asp38 by His had no effect on the uptake. All the variants of the Ir-embedded ferritin cages remained as stable as the wild-type analogue. These hybrid bio-nanocages of ferritin were found to efficiently catalyze transfer hydrogenation of various substituted acetophenones forming the corresponding chiral alcohols with up to 88 % conversion and 70 % enantioselectivity. An electron-withdrawing substituent on the reactant enhanced the Turnover frequency of the reaction. Molecular docking analyses suggested that the substrate binds in different orientations at the active site in different mutants of the nanocage.

An Individual-Level Meta-Analysis Using Real-World and Pivotal Studies on Mortality From the Use of Paclitaxel-Containing Devices in Japanese Femoropopliteal Disease Patients.

BACKGROUND: The effect of treatment with paclitaxel-containing devices (PTXD) on mortality in patients with peripheral artery disease remains controversial.Methods and Results:An independent patient-level meta-analysis of 12 clinical trials (1,389 PTXD patients and 1,192 non-PTXD patients) was conducted. This study included 7 pivotal trials and 5 post-marketing surveillance studies on endovascular treatment for femoropopliteal artery by 6 companies. The primary endpoint was all-cause death, and 5-year cumulative mortality was estimated by a Kaplan-Meier curve. Cox proportional hazard model was used to calculate the hazard ratio (HR) and confidential interval (CI). During the median follow up of 3.0 years, 459 patients died. The cumulative 5-year mortality for the entire cohort was significantly lower in the PTXD than in the non-PTXD group (24.4% vs. 27.4%, respectively; HR, 0.81; 95% CI, 0.67-0.97; P=0.023), but this difference was no longer significant after adjustment for relevant covariates (HR, 1.01; 95% CI, 0.39-2.58; P=0.987). The Cox proportional hazard model revealed that sex, hyperlipidemia, Type 2 diabetes, hemodialysis, Rutherford category, and age above 75 years were significantly associated with 5-year mortality, but treatment with PTXD was not. CONCLUSIONS: This large individual meta-analysis of patients with femoropopliteal artery disease found that the use of PTXD does not have a negative effect on 5-year mortality.

Single Antiplatelet Therapy With Prasugrel vs. Dual Antiplatelet Therapy in Japanese Percutaneous Coronary Intervention Patients With High Bleeding Risk.

BACKGROUND: Outcomes with prasugrel single antiplatelet therapy (SAPT) vs. dual antiplatelet therapy (DAPT) in Japanese percutaneous coronary intervention (PCI) patients with high bleeding risk (HBR) are currently unknown.Methods and Results:Data from 1,173 SAPT and 2,535 DAPT patients from the PENDULUM mono and PENDULUM registry studies (respective median DAPT durations: 108 vs. 312 days) were compared. The adjusted cumulative incidence of Bleeding Academic Research Consortium (BARC) 2, 3, or 5 bleeding from 1 to 12 months after PCI (primary endpoint) was 2.8% (95% confidence interval [CI], 1.9-4.2) and 4.1% (95% CI, 3.3-5.1), respectively (hazard ratio [HR], 0.69; 95% CI, 0.45-1.06; P=0.090). The adjusted cumulative incidences of BARC 2, 3, or 5 bleeding from 0 to 12 months after PCI (secondary endpoint) were 3.8% (95% CI, 2.7-5.3) and 5.6% (95% CI, 4.7-6.7), respectively (HR, 0.68; 95% CI, 0.47-0.98; P=0.039). There was no significant difference in major adverse cardiac and cerebrovascular events (MACCE) from 1 to 12 months after PCI (HR, 0.93; 95% CI, 0.63-1.37; P=0.696) and at 12 months after PCI (HR, 0.85; 95% CI, 0.61-1.19; P=0.348) between the groups. CONCLUSIONS: Prasugrel SAPT may reduce BARC 2, 3, or 5 bleeding, without increasing MACCE, in Japanese patients with HBR.

Successful introduction of robotic-assisted percutaneous coronary intervention system into Japanese clinical practice: a first-year survey at single center.

In Japan, a robotic-assisted PCI (R-PCI) system, the CorPath GRX System (Corindus Inc.), has been approved for clinical use in 2018, which is the first introduction of R-PCI into Japan. In this study, the clinical performance of the R-PCI system in the initial year at Kurume University Hospital was evaluated comparing with conventional manual PCI (M-PCI). A total of 30 R-PCI and 77 M-PCI procedures performed between April 2019 and March 2020, were retrospectively included. The primary outcome was the rate of clinical success defined as < 30% residual stenosis without in-hospital major adverse cardiovascular events (MACE). The secondary outcomes were fluoroscopy time, dose area product (DAP), amount of radiation exposure to operators and assistants, procedural time, and contrast volume. Propensity-matching technique was used to match each R-PCI lesion to the nearest M-PCI lesion without replacement. After propensity score matching, 30 R-PCI procedures in 28 patients and 37 M-PCI procedures in 35 patients were analyzed. Clinical success rate with R-PCI was favorable and comparable to M-PCI (93.3 vs. 94.6%, p = 0.97), without any in-hospital MACE. The operator radiation exposure was significantly lower in R-PCI (0 vs. 24.5 µSV, p < 0.0001). Radiation exposure to the patients was tended to be reduced by R-PCI (DAP: 77.6 vs. 100.2 Gycm2, p = 0.07). There were no statistically significant differences in radiation exposure to the assistant, fluoroscopy time, procedural time and contrast volume between the two groups (radiation exposure to the assistant: 10.5 vs. 10.0 µSV, p = 0.64, fluoroscopy time: 27.5 vs. 30.1 min, p = 0.55, procedural time: 72.4 vs. 61.6 min, p = 0.23, and contrast volume: 93.2 vs. 102.0 ml, p = 0.36). R-PCI in selected patients demonstrated favorable clinical outcomes with dramatical reduction of radiation exposure to operators.

The Versatile Manipulations of Self-Assembled Proteins in Vaccine Design.

Protein assemblies provide unique structural features which make them useful as carrier molecules in biomedical and chemical science. Protein assemblies can accommodate a variety of organic, inorganic and biological molecules such as small proteins and peptides and have been used in development of subunit vaccines via display parts of viral pathogens or antigens. Such subunit vaccines are much safer than traditional vaccines based on inactivated pathogens which are more likely to produce side-effects. Therefore, to tackle a pandemic and rapidly produce safer and more effective subunit vaccines based on protein assemblies, it is necessary to understand the basic structural features which drive protein self-assembly and functionalization of portions of pathogens. This review highlights recent developments and future perspectives in production of non-viral protein assemblies with essential structural features of subunit vaccines.