An integrated computational pipeline for designing high-affinity nanobodies with expanded genetic codes.

Protein engineering and design principles employing the 20 standard amino acids have been extensively used to achieve stable protein scaffolds and deliver their specific activities. Although this confers some advantages, it often restricts the sequence, chemical space, and ultimately the functional diversity of proteins. Moreover, although site-specific incorporation of non-natural amino acids (nnAAs) has been proven to be a valuable strategy in protein engineering and therapeutics development, its utility in the affinity-maturation of nanobodies is not fully explored. Besides, current experimental methods do not routinely employ nnAAs due to their enormous library size and infinite combinations. To address this, we have developed an integrated computational pipeline employing structure-based protein design methodologies, molecular dynamics simulations and free energy calculations, for the binding affinity prediction of an nnAA-incorporated nanobody toward its target and selection of potent binders. We show that by incorporating halogenated tyrosines, the affinity of 9G8 nanobody can be improved toward epidermal growth factor receptor (EGFR), a crucial cancer target. Surface plasmon resonance (SPR) assays showed that the binding of several 3-chloro-l-tyrosine (3MY)-incorporated nanobodies were improved up to 6-fold into a picomolar range, and the computationally estimated binding affinities shared a Pearson's r of 0.87 with SPR results. The improved affinity was found to be due to enhanced van der Waals interactions of key 3MY-proximate nanobody residues with EGFR, and an overall increase in the nanobody's structural stability. In conclusion, we show that our method can facilitate screening large libraries and predict potent site-specific nnAA-incorporated nanobody binders against crucial disease-targets.

[Distal Aortic Arch Aneurysm, Acute type B Aortic Dissection, and Acute Bilateral Limb Ischemia Treated by Two-stage Total Arch Replacement;Report of a Case].

A 74-year-old female patient experienced sudden and severe pain in her lower back and both legs. Upon examination, her femoral pulses were weak, and her legs were pale. Computed tomography revealed a 66-mm thoracic aneurysm in the distal arch and type B aortic dissection. Stenosis was present from the terminal aorta to the iliac arteries. The left common iliac artery was occluded at its bifurcation, and both lower limbs were ischemic. We performed bilateral axillary-femoral artery bypass, which improved blood flow to both limbs. The next day, it was apparent that compartment syndrome had developed in the patient's left leg. Rehabilitation therapy was effective for the compartment syndrome, the patient's symptoms resolved, and she was discharged. We later performed two-stage total arch replacement, and the subsequent clinical course has been without incident.

Perfusion through the dorsalis pedis artery for acute limb ischemia secondary to an occlusive arterial cannula during percutaneous cardiopulmonary support.

Percutaneous cardiopulmonary support (PCPS) is a powerful resuscitation tool for patients in cardiogenic shock. The femoral artery is generally used for arterial access; however, vascular complications, particularly in atherosclerotic arteries, can occur. Although such complications occur infrequently, they can be fatal. We describe the case of a 75-year-old woman who required extended PCPS for cardiogenic shock secondary to coronary spasm after on-pump beating coronary artery bypass grafting. Limb ischemia occurred because of an occlusive cannula, and distal perfusion with a 20G elastic intravenous catheter inserted into the dorsalis pedis artery resolved the ischemia. The catheter was connected to the side port of an oxygenator and provided distal limb perfusion during PCPS. This technique appears to be useful in treating limb ischemia and may have application in patients with arterial occlusive disease who are dependent on mechanical support.