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.

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.

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.

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.

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.

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.

Self-contained image mapping of placental vasculature in 3D ultrasound-guided fetoscopy.

BACKGROUND: Surgical navigation technology directed at fetoscopic procedures is relatively underdeveloped compared with other forms of endoscopy. The narrow fetoscopic field of views and the vast vascular network on the placenta make examination and photocoagulation treatment of twin-to-twin transfusion syndrome challenging. Though ultrasonography is used for intraoperative guidance, its navigational ability is not fully exploited. This work aims to integrate 3D ultrasound imaging and endoscopic vision seamlessly for placental vasculature mapping through a self-contained framework without external navigational devices. METHODS: This is achieved through development, integration, and experimentation of novel navigational modules. Firstly, a framework design that addresses the current limitations based on identified gaps is conceptualized. Secondly, integration of navigational modules including (1) ultrasound-based localization, (2) image alignment, and (3) vision-based tracking to update the scene texture map is implemented. This updated texture map is projected to an ultrasound-constructed 3D model for photorealistic texturing of the 3D scene creating a panoramic view of the moving fetoscope. In addition, a collaborative scheme for the integration of the modular workflow system is proposed to schedule updates in a systematic fashion. Finally, experiments are carried out to evaluate each modular variation and an integrated collaborative scheme of the framework. RESULTS: The modules and the collaborative scheme are evaluated through a series of phantom experiments with controlled trajectories for repeatability. The collaborative framework demonstrated the best accuracy (5.2 % RMS error) compared with all the three single-module variations during the experiment. Validation on an ex vivo monkey placenta shows visual continuity of the freehand fetoscopic panorama. CONCLUSIONS: The proposed developed collaborative framework and the evaluation study of the framework variations provide analytical insights for effective integration of ultrasonography and endoscopy. This contributes to the development of navigation techniques in fetoscopic procedures and can potentially be extended to other applications in intraoperative imaging.

A New Suturing Device for Small Arteries.

Endoscope-assisted surgery and robot-assisted surgery are not common in cardiac surgery, particularly coronary artery bypass grafting, because of the complex nature of the procedures. We developed a new suturing device that allows for easy performance of such cardiac surgeries in comparison with conventional suturing methods. A total of 63 rabbits were used in this study. The right carotid artery was bypassed using the same side of the jugular vein under endoscopic guidance. Of these, 48 rabbits were operated on using the new devices and 15 rabbits were operated on using conventional polypropylene sutures. The proximal suturing time was 16.6 ± 5.3 minutes in the group that underwent surgery using the new device (group D) and 22.8 ± 7.6 minutes in the control group (group C; P < 0.05). The distal suture time was 16.3 ± 4.2 minutes in group D and 22.8 ± 6.0 minutes in group C (P < 0.05). The operation time was 113.0 ± 15.8 minutes in group D and 136.7 ± 20.6 minutes in group C (P < 0.05). Graft flow was 19.9 ± 12.8 mL/minute in group D and 12.1 ± 11.3 mL/minute in group C (P < 0.05). Thus, the operation time and the suture time differed significantly between the groups. This device provides advantages in endoscopic surgery compared to the conventional suture method.

Development of a suturing device for anastomosis for small caliber arteries.

The use of minimally invasive and robotic operations has been increasing for coronary artery bypass surgery; however, no suturing devices have been universally adopted for use in these procedures. We developed a new suturing device that enables omission of manual ligation after a running suture. Twenty-two rabbits were used in this study. In 22 rabbits, the right carotid artery was bypassed using an autologous jugular vein graft. Half of the animals were operated on using the new device and the other half using conventional suturing methods. Postoperative evaluations were performed at 1, 3, and 6 months. Suturing time was 15.6 ± 2.4 min in the device group and 16.6 ± 4.4 min in the control group (p = 0.34). Graft patency and blood flow measurements were not significantly different between the two groups. Histopathological examination of the anastomotic site showed common inflammatory responses in both groups. No particular histopathological change was seen related to the device. In conclusion, the safety of the new suturing device was confirmed, and its efficacy was equal to that of the conventional suturing technique.

Automatic laser scanning ablation system for high-precision treatment of brain tumors.

Complete removal of malignant gliomas is important for the prognosis in neurosurgery treatment. Currently, the challenge is how to detect any remaining tumors and resect them during the operation. We have developed a laser ablation system with accurate tumor analysis and fluorescence guidance for high-precision brain tumor resection during neurosurgery. A 5-aminolevulinic acid-induced fluorescent protoporphyrins IX (PpIX)-based intra-operative fluorescence measurement and corresponding spectra analysis technique is used to identify the position of tumors. A galvano mirror scanning mechanism is integrated into the fluorescence measurement and the laser ablation devices for automatic tumor area scanning and corresponding laser ablation. A set of phantom experiments was performed to evaluate the proposed system. Results showed that the galvano scanning mechanism enabled both PpIX fluorescence detection and laser ablation in the same optical axis. In vitro experiments using porcine brain were performed to evaluate the effectiveness of the automatic laser scanning, fluorescence detection, and laser ablation system. The proposed fluorescence-guided laser ablation system can provide accurate analysis and high-precision treatment for tumor resection in neurosurgery. With further improvement, the system can be used in neurosurgical implementation to provide accurate, safe, and simple surgical diagnosis and therapy.

Direct minimally invasive intraoperative electrophysiological mapping of the heart.

INTRODUCTION: Cardiac electrophysiology aims to describe and treat the electrical activity of the heart. Although an epicardial approach is valuable in many surgical treatments such as coronary artery bypass grafting, maze ablation, and cell transplantation, very few techniques suited for minimally invasive surgery are available for measurement of epicardial electrophysiology. MATERIAL AND METHODS: We developed a novel endoscopically-deployable expanding electrode array that can be applied for minimally invasive surgery. Our device consists of a flexible electrode array attached to arms which open and close the electrode sheet. Furthermore, we also developed a computer program to overlay an epicardial electrophysiological map on an endoscopic image. We performed both laboratory and in vivo experiments to examine the feasibility in clinical situations. RESULTS: Evaluation experiments demonstrated that our novel mapping process that assumes spherical deformation of the electrode array enables us to overlay each electrode position with an accuracy of < 1 mm. Results of animal experiments using large animals (one dog and two pigs) demonstrated that our system enables construction of epicardial electrophysiological maps. CONCLUSION: A novel endoscopically deployable expanding electrode array was developed. Evaluation experiments demonstrated that our device can be manipulated in simulated minimally invasive surgery, and enables construction of epicardial electrophysiological maps.

Cardiac variation of inferior vena cava: new concept in the evaluation of intravascular blood volume.

Evaluation of the intravascular blood volume is an important assessment in emergency and critical care medicine. Measurement of the inferior vena cava (IVC) respiratory variation by ultrasound echography is useful, but it entails subjective problems. We have hypothesized that IVC cardiac variation is also correlated with intravascular blood volume and analyzed it automatically using computer software of two kinds, later comparing the results. Snakes, software to track boundaries by curve line continuity, and template matching software were incorporated into a computer with an ultrasound machine to track the short-axis view of IVC automatically and analyze it with approximation by ellipse. Eight healthy volunteers with temporary mild hypovolemia underwent echography before and after passive leg raising and while wearing medical anti-shock trousers. IVC cardiac variation was visually decreased by both leg raising and medical anti-shock trousers. The collapse index (maximum - minimum/maximum) of area during three cardiac beats was decreased showing a good relationship to fluid load simulations; 0.24 ± 0.03 at baseline versus 0.11 ± 0.01 with leg raising and 0.12 ± 0.01 with medical anti-shock trousers. In conclusion, IVC cardiac variation has the potential to provide an evaluation of water volume. It presents some advantages in mechanical analysis over respiratory variation. At the very least, we need to exercise some caution with cardiac variation when evaluating respiratory variation.

In vitro display evolution of unnatural peptides spontaneously cyclized via intramolecular nucleophilic aromatic substitutions.

We report novel, ribosomally incorporatable, and intramolecularly cysteine-reactive fluorobenzoic acid-derived linkers for SELEX of mRNA-displayed unnatural peptides, which spontaneously cyclized via intramolecular nucleophilic aromatic substitutions forming thioethers. With this strategy we identified several novel PCSK9-binding peptides.

In vitro selection generates RNA aptamer that antagonizes PCSK9-LDLR interaction and recovers cellular LDL uptake.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) induces low-density lipoprotein (LDL)-receptor (LDLR) degradation, increasing plasma LDL-cholesterol levels and causing hypercholesterolemia. Therefore, inhibition of PCSK9-LDLR interaction is an attractive therapeutic target for hypercholesterolemia treatment. In this study, we have identified a novel RNA aptamer that binds specifically to PCSK9 by in vitro selection, also known as systematic evolution of ligands by exponential enrichment (SELEX). The binding kinetics of the PCSK9-binding RNA aptamer was measured by biolayer interferometry assay, showing that the aptamer has higher affinity compared to PCSK9-LDLR interaction. Competitive inhibition assay using chemiluminescence detection revealed that the RNA aptamer inhibits PCSK9-LDLR interaction. In cellular LDL-uptake assays with HepG2 cells, the RNA aptamer recovered LDL uptake in the PCSK9-treated cells, demonstrating its anti-PCSK9 antagonistic activity. These results indicated that the PCSK9-binding RNA aptamer has the potential to be an affinity reagent for PCSK9 protein analysis and a therapeutic reagent for hypercholesterolemia treatment.

Novel Anastomotic Device for Distal Coronary Anastomosis: Preclinical Results From Swine Off-Pump Coronary Artery Bypass Model.

PURPOSE: We evaluated the safety and feasibility of a new anastomotic device that simplifies coronary distal anastomosis during minimally invasive and robotically assisted coronary artery bypass graft surgery (CABG). DESCRIPTION: Fourteen miniature pigs underwent off-pump CABG using bilateral internal thoracic arteries (ITA), namely, left ITA to left anterior descending artery and right ITA to right coronary artery. The device was used for distal anastomosis in a device group (n = 11), and conventional 7-0 polypropylene suture in a control group (n = 3). Graft flow was measured intraoperatively. One-month, 3-month, and 6-month postoperative angiography evaluations were done. Histopathologic examination of the anastomosis was also done. EVALUATION: Baseline and intraoperative characteristics were similar in the two groups. There was no difference of anastomotic time between groups (p = 0.59). Graft flows were also similar (p = 0.55), with good diastolic pattern in both groups. Angiography demonstrated FitzGibbon A patency in all anastomoses of both groups at each evaluation period. Histopathologic examination showed nonspecific inflammatory changes in the device group. CONCLUSIONS: The safety and feasibility of this anastomotic device for distal coronary anastomosis were shown in the swine model.

Towards scene adaptive image correspondence for placental vasculature mosaic in computer assisted fetoscopic procedures.

BACKGROUND: Visualization of the vast placental vasculature is crucial in fetoscopic laser photocoagulation for twin-to-twin transfusion syndrome treatment. However, vasculature mosaic is challenging due to the fluctuating imaging conditions during fetoscopic surgery. METHOD: A scene adaptive feature-based approach for image correspondence in free-hand endoscopic placental video is proposed. It contributes towards existing techniques by introducing a failure detection method based on statistical attributes of the feature distribution, and an updating mechanism that self-tunes parameters to recover from registration failures. RESULTS: Validations on endoscopic image sequences of a phantom and a monkey placenta are carried out to demonstrate mismatch recovery. In two 100-frame sequences, automatic self-tuned results improved by 8% compared with manual experience-based tuning and a slight 2.5% deterioration against exhaustive tuning (gold standard). CONCLUSION: This scene-adaptive image correspondence approach, which is not restricted to a set of generalized parameters, is suitable for applications associated with dynamically changing imaging conditions. Copyright © 2015 John Wiley & Sons, Ltd.

Cardiac Variation of Internal Jugular Vein for the Evaluation of Hemodynamics.

Evaluations of intravascular fluid volume are considered to be one of the most important assessments in emergency and intensive care. Focusing on pulse-induced variation of the internal jugular vein (IJV) area, i.e., cardiac variation, we investigated its correlation with various hemodynamic indices using newly developed software. Software that automatically can track and analyze the IJV during ultrasonography was developed. Eleven healthy patients were subjected to an exercise load to increase their stroke volume (SV) and a dehydration load to decrease their central venous pressure (CVP). The cardiac variation in the area of the IJV, CVP, the SV and the respiratory variation in the inferior vena cava (IVC) were evaluated. The exercise protocol increased the patients' mean SV by 14.5 ± 3.7 mL, and the dehydration protocol caused their mean CVP to fall by 3.75 ± 0.33 cm H2O, which resulted in the collapse index (max IJV area - min IJV area/max IJV area) changing from 0.32 ± 0.04 to 0.44 ± 0.06 and 0.49 ± 0.04, respectively (p < 0.05). The SV exhibited a strong positive correlation with the collapse index (r = 0.59, p = 0.006), and CVP showed a strong positive correlation with the body height-adjusted mean area of the IJV (r = 0.72, p < 0.001). Cardiac variation in the area of the great veins is considered to be induced by venous return to the right atrium under negative pressure. It is possible that intravascular dehydration can be detected and hemodynamic indices, such as CVP and SV, can be estimated by evaluating cardiac variation in the area of the IJV.

Localizing fluorophore (centroid) inside a scattering medium by depth perturbation.

Fluorescence molecular tomography (FMT) imaging can be used to determine the location, size, and biodistribution of fluorophore biomarkers inside tissues. Yet when using FMT in the reflectance geometry it is challenging to accurately localize fluorophores. A depth perturbation method is proposed to determine the centroid of fluorophore inside a tissue-like medium. Through superposition of a known thin optical phantom onto the medium surface, the fluorophore depth is deliberately perturbed and signal localization is improved in a stable way. We hypothesize that the fluorophore centroid can be better localized through use of this fluorescent intensity variation resulting from the depth perturbation. This hypothesis was tested in tissue-like phantoms. The results show that a small-size fluorophore inclusion (1.2 mm(3)volume, depth up to 4.8 mm) can be localized by the method with an error of 0.2 to 0.3 mm. The method is also proven to be capable of handling multiple fluorescent inclusion conditions with the assistance of other strategies. Additionally, our further studies showed that the method's performance in the presence of background fluorophores indicated that the small inclusion could be located at a 1.8 (3.8) mm depth with accurate localization only when its concentration was not <10 (100) times the background level.