SELEX-discovered aptamer that inhibits cellular interleukin-17/interleukin-17 receptor interaction and antagonizes interleukin-17 signaling.

This research is based on a Systematic Evolution of Ligands by EXponential enrichment, also referred to as in vitro selection against the extracellular domain of human interleukin-17 receptor A (IL-17RA). Pull-down assay via quantitative polymerase chain reaction and chemiluminescence detection showed that the cloned RNA with the enriched sequence bound to human IL-17RA and inhibited the interaction between IL-17RA and human interleukin-17A (IL-17A). We also revealed that the newly discovered IL-17RA-binding RNA aptamer bound to cellular IL-17RA, inhibited the cellular IL-17RA/IL-17A interaction, and antagonized cellular IL-17A signaling.

Theoretical elucidation of the structure, bonding, and reactivity of the CaMn4Ox clusters in the whole Kok cycle for water oxidation embedded in the oxygen evolving center of photosystem II. New molecular and quantum insights into the mechanism of the O-O bond formation.

This paper reviews our historical developments of broken-symmetry (BS) and beyond BS methods that are applicable for theoretical investigations of metalloenzymes such as OEC in PSII. The BS hybrid DFT (HDFT) calculations starting from high-resolution (HR) XRD structure in the most stable S1 state have been performed to elucidate structure and bonding of whole possible intermediates of the CaMn4Ox cluster (1) in the Si (i = 0 ~ 4) states of the Kok cycle. The large-scale HDFT/MM computations starting from HR XRD have been performed to elucidate biomolecular system structures which are crucial for examination of possible water inlet and proton release pathways for water oxidation in OEC of PSII. DLPNO CCSD(T0) computations have been performed for elucidation of scope and reliability of relative energies among the intermediates by HDFT. These computations combined with EXAFS, XRD, XFEL, and EPR experimental results have elucidated the structure, bonding, and reactivity of the key intermediates, which are indispensable for understanding and explanation of the mechanism of water oxidation in OEC of PSII. Interplay between theory and experiments have elucidated important roles of four degrees of freedom, spin, charge, orbital, and nuclear motion for understanding and explanation of the chemical reactivity of 1 embedded in protein matrix, indicating the participations of the Ca(H2O)n ion and tyrosine(Yz)-O radical as a one-electron acceptor for the O-O bond formation. The Ca-assisted Yz-coupled O-O bond formation mechanisms for water oxidation are consistent with recent XES and very recent time-resolved SFX XFEL and FTIR results.

Model calculations for the prediction of the diradical character of physisorbed molecules: p-benzyne/MgO and p-benzyne/SrO.

The analysis of the diradical state of functional open-shell molecules is important for understanding their physical properties and chemical reactivity. The diradical character is an important factor in the functional elucidation and design of open-shell molecules. In recent years, attempts have been made to immobilise functional open-shell molecules on surfaces to form devices. However, the influence of surface interactions on the diradical state remains unclear. In this study, the physisorption structures of p-benzyne, which is a typical diradical molecule, on MgO(001) and SrO(001) surfaces are used as models to investigate how the diradical character is affected by physisorption. This is done using approximate spin-projected density functional theory calculations with dispersion correction and plane-wave basis (AP-DFT-D3/plane-wave calculations). The diradical character change (Δy) due to adsorption can be categorised into three factors, namely the change due to the distortion of the diradical molecule (Δydis), the interaction between neighbouring diradical molecules (Δycoh), and molecule-surface interactions (Δysurf). In all the calculated models, physisorption reduced the diradical character (Δy < 0), and the contribution of Δysurf was the largest among the three factors. The calculated results show that adsorption induces electron delocalisation to π-conjugated orbitals and intramolecular charge polarisation, both of which contribute to reducing the occupancy of singly occupied molecular orbitals. This indicates that the diradical character of p-benzyne is reduced by the stabilisation of the resonance structures. Furthermore, geometry optimisation of the surfaces shows that the chemical-soft surface (SrO) varies the diradical character more significantly than the chemical-hard surface (MgO). This study shows that the open-shell electronic state and stack structure of diradical molecules can be controlled through the analysis of the surface diradical state.

The Nature of the Chemical Bonds of High-Valent Transition-Metal Oxo (M=O) and Peroxo (MOO) Compounds: A Historical Perspective of the Metal Oxyl-Radical Character by the Classical to Quantum Computations.

This review article describes a historical perspective of elucidation of the nature of the chemical bonds of the high-valent transition metal oxo (M=O) and peroxo (M-O-O) compounds in chemistry and biology. The basic concepts and theoretical backgrounds of the broken-symmetry (BS) method are revisited to explain orbital symmetry conservation and orbital symmetry breaking for the theoretical characterization of four different mechanisms of chemical reactions. Beyond BS methods using the natural orbitals (UNO) of the BS solutions, such as UNO CI (CC), are also revisited for the elucidation of the scope and applicability of the BS methods. Several chemical indices have been derived as the conceptual bridges between the BS and beyond BS methods. The BS molecular orbital models have been employed to explain the metal oxyl-radical character of the M=O and M-O-O bonds, which respond to their radical reactivity. The isolobal and isospin analogy between carbonyl oxide R2C-O-O and metal peroxide LFe-O-O has been applied to understand and explain the chameleonic chemical reactivity of these compounds. The isolobal and isospin analogy among Fe=O, O=O, and O have also provided the triplet atomic oxygen (3O) model for non-heme Fe(IV)=O species with strong radical reactivity. The chameleonic reactivity of the compounds I (Cpd I) and II (Cpd II) is also explained by this analogy. The early proposals obtained by these theoretical models have been examined based on recent computational results by hybrid DFT (UHDFT), DLPNO CCSD(T0), CASPT2, and UNO CI (CC) methods and quantum computing (QC).

Discovery of IL-5-binding unnatural cyclic peptides from multiple libraries by directed evolution.

Interleukin-5 (IL-5) is a type 2 cytokine involved in various allergic diseases, including severe eosinophilic asthma. In this study, we performed directed evolution against human IL-5 using systematic evolution of ligands by exponential enrichment (SELEX) from multiple mRNA-displayed peptide libraries. Peptide libraries were prepared with Escherichia coli-based reconstituted cell-free transcription and translation coupling system (PURE system) and spontaneously cyclized using multiple intramolecularly thiol-reactive benzoic acid-derived linkers, which were ribosomally incorporated through genetic code expansion. We successfully identified multiple novel IL-5-binding unnatural cyclic peptides with different cyclization linkers from multiple highly diverse mRNA-displayed libraries. Chemical dimerization was also performed to increase the avidity of unnatural cyclic IL-5-binding peptides. The novel IL-5-binding unnatural cyclic peptides discovered in this study could be used in various research, therapeutic, and diagnostic applications involving IL-5 signaling.

Artificial aptamer that inhibits interleukin-23/interleukin-23 receptor interaction discovered via SELEX.

Interaction between the pro-inflammatory cytokine interleukin-23 (IL-23) and IL-23 receptor (IL-23R) is related to the development of inflammatory autoimmune diseases such as psoriasis, inflammatory bowel disease, and Crohn's disease. In this study, we conducted systematic evolution of ligands by exponential enrichment (SELEX) for in vitro selection against human IL-23 and observed RNA sequence enrichment in the final SELEX round. IL-23-pull-down assay by chemiluminescence detection and fluorescence imaging demonstrated that SELEX-enriched RNA clone bound to IL-23. Quantitative polymerase chain reaction-based pull-down assay using the IL-23 alpha (IL-23A) subunit, a component of the IL-23 heterodimer, indicated that the RNA clone bound to IL-23A, which is favorable for autoimmune disease treatment. We also observed that the novel IL-23-binding RNA aptamer inhibited interaction between IL-23 and IL-23R. Thus, the novel IL-23-binding RNA aptamer can be used for IL-23 studies and has potential to be used for IL-23 diagnosis and IL-23-related inflammatory autoimmune disease treatment.

A human epidermal growth factor receptor 3/heregulin interaction inhibitor aptamer discovered using SELEX.

The interaction of human epidermal growth factor receptor 3 (HER3) and heregulin (HRG) is involved in resistance to human epidermal growth factor receptor 2 (HER2)-targeted cancer treatment, such as therapies using anti-HER2 monoclonal antibody. Therefore, inhibition of the HER3/HRG interaction is potentially valuable therapeutic target for cancer treatment. In this study, we used in vitro selection, also known as systematic evolution of ligands by exponential enrichment (SELEX) against the extracellular domain of human HER3, and discovered a novel RNA aptamer. Pull-down and bio-layer interferometry assays showed that RNA aptamer discovered specifically bound to HER3 with a dissociation constant (KD) of 700 nM. Pull-down assays using chemiluminescence detection also revealed that the HER3-binding RNA aptamer inhibited interactions between HER3 and human HRG. These results indicated that the novel HER3-binding RNA aptamer has potential to be used as basic tool in a range of applications involving HER3/HRG interactions, including research, therapeutic, and diagnostic applications.

In vitro display evolution of IL-6R-binding unnatural peptides ribosomally initiated and cyclized with m-(chloromethyl)benzoic acid.

The interaction of the multifunctional cytokine interleukin (IL)-6 and its receptor (IL-6R) is involved in various diseases, including not only autoimmune diseases such as rheumatoid arthritis but also cancer and cytokine storms in coronavirus disease 2019 (COVID-19). In this study, systematic evolution of ligands by exponential enrichment (SELEX) against human IL-6R from mRNA-displayed unnatural peptide library ribosomally initiated and cyclized with m-(chloromethyl)benzoic acid (mClPh) incorporated by genetic code expansion (sense suppression) was performed using the PURE (Protein synthesis Using Recombinant Elements) system. A novel 13-mer unnatural mClPh-cyclized peptide that binds to the extracellular domain of IL-6R was discovered from an extremely diverse random peptide library. In vitro affinity maturation of IL-6R-binding unnatural mClPh-cyclized peptide from focused libraries was performed, identifying two IL-6R-binding unnatural mClPh-cyclized peptides by next-generation sequencing. Because cyclization can increase the protease resistance of peptides, novel IL-6R-binding mClPh-cyclized peptides discovered in this study have the potential to be used for a variety of research, therapeutic, and diagnostic applications involving IL-6/IL-6R signaling.

Directed evolution of dibenzocyclooctyne-reactive peptide tags for protein labeling.

Protein labeling with a functional molecule is a technique widely used for protein research. The covalent reaction of self-labeling peptide tags with synthetic probe-modified small molecules enables tag-fused protein labeling with chemically diverse molecules, including fluorescent probes. We report the discovery, by in vitro directed evolution, of a novel 23-mer dibenzocyclooctyne (DBCO)-reactive peptide (DRP) tag using Systematic Evolution of Ligands by EXponential enrichment (SELEX) with a combination of a reconstituted cell-free translation system (PURE system) and cDNA display. The N- and C-terminal DRP truncations created a shorter 16-mer DBCO-reactive peptide (sDRP) tag without significant reactivity reduction. By fusing the sDRP tag to a model protein, we showed the chemical labeling and in-gel fluorescence imaging of the sDRP-fused protein using a fluorescent DBCO probe. Results showed that sDRP tag-mediated protein labeling has potential for use as a basic molecular tool in a variety of applications for protein research.

In vitro display evolution of the PURE system-expressed TNFα-binding unnatural cyclic peptide containing an N-methyl-d-amino acid.

Tumor necrosis factor-alpha (TNFα) is a multifunctional cytokine associated with inflammation, immune responses, and autoimmune diseases including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. In the present study, we performed in vitro selection, systematic evolution of ligands by exponential enrichment (SELEX) against human TNFα from mRNA-displayed peptide library prepared with Escherichia coli-reconstituted cell-free transcription/translation system (PURE system) and cyclized by N-chloroacetyl-N-methyl-d-phenylalanine incorporated by genetic code expansion (sense suppression). We identified a novel TNFα-binding thioether-cyclized peptide that contains an N-methyl-d-phenylalanine. Since cyclic structure and presence of an N-methyl-d-amino acid can increase proteolytic stability, the TNFα binding peptide has potential to be used for therapeutic, research and diagnostic applications.

Treatment effect prediction using CT after balloon pulmonary angioplasty in chronic thromboembolic pulmonary hypertension.

OBJECTIVE: To evaluate whether the change in computed tomography pulmonary angiography (CTPA) metrics after balloon pulmonary angioplasty (BPA) can predict treatment effect in chronic thromboembolic pulmonary hypertension (CTEPH) patients. METHODS: This study included 82 CTEPH patients who underwent both CTPA and right heart catheterization (RHC) before and at the scheduled time of 6 months after BPA. The diameters of the main pulmonary artery (dPA), ascending aorta (dAA), right atrium (dRA), right ventricular free wall thickness (dRVW), and right and left ventricles (dRV, dLV) were measured on CTPA. The correlation of the New York Heart Association functional class (NYHA FC), 6-minute walking distance (6MWD), brain natriuretic peptide (BNP) level, and calculated CT metrics with a decrease in mean pulmonary artery pressure (ΔmPAP) using RHC (used as the reference for BPA effect) was investigated. Using multiple regression analysis, independent variables were also identified. RESULTS: In univariate analysis, clinical indicators (NYHA FC, 6MWD, and BNP level) improved significantly after BPA and were significantly correlated with ΔmPAP (p < 0.01). In the univariate analysis of CTPA parameters, dPA, dRA, dPA/dAA ratio, dRVW, and dRV/dLV ratio decreased significantly and were significantly correlated with ΔmPAP (p < 0.01). Multivariate analysis demonstrated that decreased dPA (p = 0.001) and decreased dRA (p = 0.039) on CTPA were independent predictive factors of ΔmPAP. CONCLUSIONS: Decreased dPA and dRA on CTPA could predict a decrease in mPAP after BPA, thus potentially eliminating unnecessary invasive catheterization. KEY POINTS: • The reduction in mean pulmonary artery pressure after balloon pulmonary angioplasty in CTEPH patients was significantly correlated with the clinical indices improvement and CTPA parameter decrease. • The decreased diameter of the main pulmonary artery and the decreased diameter of the right atrium on CTPA were independent predictors of mean pulmonary artery pressure reduction.

Clinical Significance of Guanylate Cyclase Stimulator, Riociguat, on Right Ventricular Functional Improvement in Patients with Pulmonary Hypertension.

BACKGROUND: Riociguat is a soluble guanylate cyclase stimulator that improves hemodynamics in patients with pulmonary hypertension (PH). Accumulating evidence implicates the additional effect of riociguat on the increase in cardiac output. However, its mechanisms have not been fully understood. This study aimed to investigate whether riociguat could ameliorate right ventricular (RV) contraction as well as hemodynamics. METHODS: We studied 45 patients with pulmonary arterial hypertension (14) or chronic thromboembolic pulmonary hypertension (31) and evaluated hemodynamics, using right-sided heart catheterization, before and after the administration of riociguat. RV function was assessed by echocardiography, including speckle-tracking echocardiography. RESULTS: Riociguat significantly improved the WHO functional class and reduced the mean pulmonary arterial pressure and vascular resistance. In addition, the cardiac index increased. RV remodeling was ameliorated after riociguat administration as assessed by the echocardiographic parameters, such as RV diameter and RV area index. RV function, including RV fractional area change and RV global longitudinal strain, also significantly improved, and their improvement was even observed in patients with mild PH after pulmonary endarterectomy or balloon pulmonary angioplasty. Furthermore, covariance analysis revealed that RV global longitudinal strain and RV fractional area change improved after riociguat administration, even with the same mean pulmonary arterial pressure, implicating the improvement of RV contractile function by riociguat, regardless of RV loading. CONCLUSIONS: Riociguat not only improved the hemodynamics of patients with PH but also ameliorated the echocardiographic parameters with RV function. RV strain could detect the subtle improvement in mild PH, and riociguat may have a benefit even after intervention, as assessed by speckle-tracking echocardiography.

Can we enhance diradical character using interaction with stoichiometric surfaces of ionic oxides? A theoretical investigation using chemical indices.

Chemical indices are effective tools for examining the functions and reactivities of stable radical species. In this study, we formulated an approximation to estimate chemical indices using electron density. Theoretical investigations using the developed scheme revealed that surface interactions can tune chemical indices and that the diradical character was enhanced by weak adsorption onto ionic solids with charge-dipole interactions.

In vitro selection of an RNA aptamer yields an interleukin-6/interleukin-6 receptor interaction inhibitor.

Interleukin-6 (IL-6) binds to the IL-6 receptor (IL-6R) subunit, related to autoimmune diseases and cytokine storm in COVID-19. In this study, we performed systematic evolution of ligands by exponential enrichment and identified a novel RNA aptamer. This RNA aptamer not only bound to IL-6R with a dissociation constant of 200 n m, but also inhibited the interaction of IL-6R with IL-6.

Predictors of New-Onset Atrial Tachyarrhythmias After Transcatheter Atrial Septal Defect Closure in Adults.

BACKGROUND: New-onset atrial tachyarrhythmia (ATA) often develops after atrial septal defect (ASD) closure. Its development raises some potential concerns such as stroke and bleeding complications caused by anticoagulant therapy and limited access to the left atrium for catheter ablation. Although it is essential to identify the risk factors of new-onset ATA, few studies have examined these factors. This study investigated unknown risk factors for the development of new-onset ATA after transcatheter ASD closure in patients without a history of ATA. METHODS: A total of 238 patients without a history of ATA, aged ≥18 years and who underwent transcatheter ASD closure at the current hospital were reviewed. Patient characteristics were compared between the groups with and without new-onset ATA. The factors associated with new-onset ATA were examined using univariate and multivariable analyses. RESULTS: Thirteen (13) (5.5%) patients experienced ATA during follow-up (mean, 21±14 months). Compared with patients without new-onset ATA, patients with new-onset ATA were older (48±18 vs 66±11 years; p<0.001) and had high brain natriuretic peptide (BNP) levels (36±36 vs 177±306 pg/mL; p<0.001). On multivariable analysis, BNP ≥40 pg/mL before ASD closure was associated with new-onset ATA after adjusting for age (OR, 4.91; 95% CI, 1.22-19.8; p=0.025). CONCLUSION: Patients with BNP levels >40 pg/mL before transcatheter ASD closure may have a higher risk of developing new-onset ATA.

Factors related to instrumental activities of daily living in persons with chronic thromboembolic pulmonary hypertension.

Instrumental activities of daily living (IADL) are significantly related to quality of life and mortality among individuals with heart disease. However, few reports have examined IADL in persons with chronic thromboembolic pulmonary hypertension (CTEPH). The aim of this study was to clarify factors related to IADL in persons with CTEPH. This retrospective, observational study enrolled 163 persons with CTEPH (mean ± standard deviation age = 65 ± 13 years; 68% female) admitted to the Department of Cardiology at Keio University Hospital between January 2015 and July 2019. The Frenchay Activities Index (FAI) was used to assess IADL. Age, sex, body mass index, World Health Organization functional class (WHO-FC), cardiac function (mean pulmonary arterial pressure, mean right atrial pressure, pulmonary capillary wedge pressure, and cardiac index), pulmonary function (percentage vital capacity, percentage forced expiratory volume in 1 s, diffusion capacity of carbon monoxide (DLCO)/alveolar volume (VA)), physical function (knee extension strength and walking speed), and 6-min walking distance (6MWD) were assessed. Multiple regression analysis was performed to identify factors significantly associated with FAI. Mean FAI was 25 ± 8. Univariate analysis showed that sex, WHO-FC, DLCO/VA, walking speed, and 6MWD were correlated with FAI. Multiple regression analysis showed that 6MWD (sß = 0.338, 95% CI 0.014-0.034, p < .001), sex (sß = 0.268, 95% CI 2.238-7.165, p < .001), and DLCO/VA (sß = 0.257, 95% CI 1.011-3.528, p < .001) were significantly correlated with FAI (R2 = 0.261). IADL were associated with exercise tolerance, sex, and DLCO/VA in persons with CTEPH. In the future, more details of IADL are expected to be clarified by analyzing individual components of IADL and investigating social background characteristics, including living environment.

Preoperative right ventricular strain predicts sustained right ventricular dysfunction after balloon pulmonary angioplasty in patients with chronic thromboembolic pulmonary hypertension.

AIMS: Balloon pulmonary angioplasty (BPA) improves hemodynamics and exercise tolerance in patients with chronic thromboembolic pulmonary hypertension (CTEPH). However, its diagnostic and predictive values remain unclear. We investigated the diagnostic and predictive values of BPA by assessing the mechanism of right ventricular (RV) dysfunction. METHODS AND RESULTS: Hemodynamic improvement was maintained over 6 months in 99 patients with CTEPH who underwent BPA. Notably, 57 of 99 patients showed normalization of pulmonary vascular resistance (PVR) after BPA. The RV mid free wall longitudinal strain (RVMFS) was inversely correlated with the 6-min walk distance (r = -.35, P = .01) and serum levels of high-sensitivity cardiac troponin T (hs-cTNT) (r = -.39, P = .004) 6 months post-BPA in the PVR-normalized group. Among all variables analyzed, only the pre-BPA RVMFS was correlated with the post-BPA RVMFS (r = .40, P = .001), and the pre-BPA RVMFS (<-15.8%) was the strongest predictor of post-BPA normalization of RVMFS (area under the curve 0.80, P = .01, sensitivity 89%, and specificity 63%). The immediate post-BPA RVMFS showed worsening over 6 months after the procedure (-25.8% to -21.1%) in patients with high serum hs-cTNT levels (>0.0014 ng/mL). In contrast, we observed an improvement in these values in those with low serum hs-cTNT levels (-23.6% to -24.4%). CONCLUSION: RVMFS of -15.8% may be a useful cutoff value to categorize the refractory and non-refractory stages of disease. Sustained serum hs-cTNT elevation post-BPA indicates subclinical RV myocardial injury, with resultant RVMFS deterioration and poor exercise tolerance.

Theoretical study of correlations between the coordination structures and catalytic activities in polymer-stabilized au nanocluster catalysts.

Au nanoclusters (Au NCs) stabilized by poly(N-vinyl-2-pyrrolidone) and poly(allylamine), abbreviated to Au:PVP and Au:PAA, catalyze the aerobic oxidation of p-hydroxybenzyl alcohols, but the catalytic activity of Au:PVP is much higher than that of Au:PAA. To elucidate the correlations between the catalytic activities and coordination structures of the stabilizing polymer, the substrate accessibility on Au NCs was estimated by density functional theory (DFT) and molecular dynamics (MD) calculations. For MD simulations, we applied a systematic method to optimize the temperature parameters in temperature replica exchange molecular dynamics (T-REMD), and the coordination structures were comprehensively classified by multivariate analysis. The results show that the number of open active sites on the Au NCs is a good index for predicting the catalytic activities. © 2018 Wiley Periodicals, Inc.

UNO DMRG CAS CI calculations of binuclear manganese complex Mn(IV)2 O2 (NHCHCO2 )4 : Scope and applicability of Heisenberg model.

Both direct exchange and super-exchange interactions cooperate to realize inter-spin magnetic interaction in binuclear manganese complex Mn(IV)2 O2 (NHCHCO2 )4 with a di-µ-oxo path. We revisited this spin system using DMRG CAS methods and CAS selection procedures. Our results indicate that our previous "dynamically extended spin polarization" (DE-SP) procedure for organic polyradicals and so forth does not work well. Thus, we have examined another selection procedure, the "dynamically extended super-exchange" (DE-SE) procedure. DMRG CASCI [18,18] by UB3LYP(HS)-UNO(DE-SE) can realize antiferromagnetic J values similar to experimental ones (-87 cm-1 ). In addition, all J values between all spin states (HS[septet],IS[quintet],IS[triplet],LS[singlet])were also shown to be correct under sufficiently large M values. © 2018 Wiley Periodicals, Inc.

Theoretical and computational investigations of geometrical, electronic and spin structures of the CaMn4 OX (X = 5, 6) cluster in the Kok cycle Si (i = 0-3) of oxygen evolving complex of photosystem II.

The optimized geometries of the CaMn4 OX (X = 5, 6) cluster in the oxygen evolving complex (OEC) of photosystem II (PSII) by large-scale quantum mechanics (QM) and molecular mechanics (MM) calculations are compared with recent serial femtosecond crystallography (SFX) results for the Si (i = 0-3) states. The valence states of four Mn ions by the QM/MM calculations are also examined in relation to the experimental results by the X-ray emission spectroscopy (XES) for the Si intermediates. Geometrical and valence structures of right-opened Mn-hydroxide, Mn-oxo and Mn-peroxide intermediates in the S3 state are investigated in detail in relation to recent SFX and XES experiments for the S3 state. Interplay between theory and experiment indicates that the Mn-oxo intermediate is a new possible candidate for the S3 state. Implications of the computational results are discussed in relation to possible mechanisms of the oxygenoxygen bond formation for water oxidation in OEC of PSII.