In vivo incorporation of phenylalanine (Phe) analogues into an artificial extracellular matrix protein (aECM-CS5-ELF) was accomplished using a bacterial expression host that harbors the mutant phenylalanyl-tRNA synthetase (PheRS) with an enlarged binding pocket. Although the Ala294Gly/Thr251Gly mutant PheRS (PheRS**) under the control of T5 promoter allows incorporation of some Phe analogues into a protein, the T5 system is not suitable for material science studies because the amount of materials produced is not sufficient due to the moderate strength of the T5 promoter. This limitation can be overcome by using a pair of T7 promoter and T7 RNA polymerase instead. In the T7 expression system, it is difficult, however, to achieve a high incorporation level of Phe analogues, due to competition of Phe analogues for incorporation with the residual Phe that is required for synthesis of active T7 RNA polymerase. In this study, we prepared the PheRS** under T7 promoter and optimized culture condition to improve both the incorporation level of recombinant aECM protein and the incorporation level of Phe analogues. Incorporation and expression levels tend to increase in the case of p-azidophenylalanine, p-iodophenylalanine, and p-acetylphenylalanine. We evaluated the lower critical transition temperature, which is dependent on the incorporation ratio and the turbidity decreased when the incorporation level increased. Circular dichromism measurement indicated that this tendency is based on conformational change from random coil to β-turn structure. We demonstrated that polyethylene glycol (PEG) can be conjugated at reaction site of Phe analogues incorporated. We also demonstrated that the increased hydrophilicity of elastin-like sequences in the aECM-CS5-ELF made by PEG conjugation could suppress nonspecific adhesion of human umbilical vein endothelial cells (HUVEC).
Objective In-stent thrombosis (IST) is a known complication after stent-assisted coil (SAC) embolization. We report a case of mechanical thrombectomy using a stent retriever (SR) for IST and share our experience with this treatment to prevent a poor outcome in future cases. Case Presentation The patient was a 62-year-old man. SAC embolization for an unruptured left internal carotid artery (ICA) aneurysm was performed. Three weeks after discharge, right hemiparesis and aphasia developed. Magnetic resonance imaging (MRI) demonstrated cerebral infarction in the left middle cerebral artery (MCA) territory and the left ICA was occluded. His relatives told us that the patient discontinued taking antiplatelet drugs. IST was diagnosed and emergency thrombectomy was performed. First, we tried to introduce an aspiration catheter or balloon catheter into the occluded lesion, but they were unable to be sufficiently inserted to the distal site. Therefore, we used a SR even though it carried a risk of friction on the deployed stent. The occluded artery was finally recanalized using the SR, but the stent became shortened. For the treatment strategy, sufficient medication (antithrombogenic agents and edaravone) should be administered first, followed by mechanical treatment. In mechanical treatment, thrombus fragmentation with a guidewire or balloon and aspiration should be attempted first. New aspiration catheters to carry the devices to the far distal site easily are now available. Conclusion SRs cannot be utilized for thrombectomy with a stent. In emergency situations, careful consideration during troubleshooting rather than using a SR is needed.
Objective: Indocyanine green videoangiography (ICG-VA) has been widely used in vascular surgery. While vessels are clearly visible as white regions on a black background, other structures cannot be observed. We developed a new system to simultaneously capture both, light and near-infrared (NIR) fluorescence images in ICG-VA (dual-image VA [DIVA]), allowing for observation of other cranial structures. We are attempting to make this technology commercially available.Materials and Methods: We used our new technology with 10 different neurovascular cases. The operative field was illuminated via an operating microscope (OPMI PENTERO 900; Carl Zeiss Meditec, Jena, Germany). In the camera unit, visible light was filtered to 400-700 nm. NIR fluorescence emission light was filtered to 800-900 nm using a special sensor unit with an optical filter. Light and NIR fluorescence images were simultaneously visualized on a single monitor.Results: All 10 cases showed a clearly visualized operative field including both fluorescence-enhanced blood vessels and other cranial structures. In aneurysm surgeries, we were able to confirm complete clipping and preserve blood flow in the parent and perforating arteries. In cases of arteriovenous malformation, feeding arteries, nearby passing arteries, and draining veins were all easily visualized and detected.Conclusions: We developed a high-resolution intraoperative imaging system to enable real-time visualization of the color operative field along with ICG fluorescence-enhanced blood flow.
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