Yoshiko Toyoda

Discovering Aptamers by Cell-SELEX against Human Soluble Growth Factors Ectopically Expressed on Yeast Cell Surface

SELEX, the process of selecting aptamers, is often hampered by the difficulty of preparing target molecules in their native forms and by a lack of a simple yet quantitative assay for monitoring enrichment and affinity of reactive aptamers. In this study, we sought to discover DNA aptamers against human serum markers for potential therapeutic and diagnostic applications. To circumvent soluble expression and immobilization for performing SELEX, we ectopically expressed soluble growth factors on the surface of yeast cells to enable cell-SELEX and devised a flow cytometry-based method to quantitatively monitor progressive enrichment of specific aptamers. High-throughput sequencing of selected pools revealed that the emergence of highly enriched sequences concurred with the increase in the percentage of reactive aptamers shown by flow cytometry. Particularly, selected DNA aptamers against VEGF were specific and of high affinity (KD = ∼ 1 nM) and demonstrated a potent inhibition of capillary tube formation of endothelial cells, comparable to the effect of a clinically approved anti-VEGF antibody drug, bevacizumab. Considering the fact that many mammalian secretory proteins have been functionally expressed in yeast, the strategy of implementing cell-SELEX and quantitative binding assay can be extended to discover aptamers against a broad array of soluble antigens.

Abstract: Efficacy and Outcomes of Fat Grafting Beyond the Breast

and improves analgesia after breast cancer surgery: a randomized, controlled, single-center trial.” PloS one 10.11 (2015): e0142249. 2. Zhong, Toni, et al. “Transversus abdominis plane (TAP) catheters inserted under direct vision in the donor site following free DIEP and MS-TRAM breast reconstruction: a prospective cohort study of 45 patients.” Journal of Plastic, Reconstructive & Aesthetic Surgery 66.3 (2013): 329–336. 3. Lou, Feifei, et al. “Epidural combined with general anesthesia versus general anesthesia alone in patients undergoing free flap breast reconstruction.” Plastic and reconstructive surgery 137.3 (2016): 502e-509e.

Toward Microsurgical Correction of Cleft Lip Ex Utero through Restoration of Craniofacial Developmental Programs

Background: Cleft lip with or without cleft palate is present in approximately one in 500 to 700 live births, representing the most common congenital craniofacial anomaly. Previously, the authors developed a unique murine model with compound Pbx deficiency that exhibits fully penetrant cleft lip with or without cleft palate. To investigate the possibility of tissue repair at an early gestational stage, the authors designed a minimally invasive surgical approach suitable for intrauterine repair using Wnt9b-soaked collagen microspheres to restore craniofacial developmental programs for cleft correction. Methods: Collagen microspheres with diameters ranging from 20 to 50 &mgr;m were fabricated to serve as a delivery vehicle for Wnt9b. At gestational day 11.5, wild-type and Pbx-deficient murine embryos were isolated. Microspheres soaked in murine purified Wnt9b protein were microsurgically implanted at the midface lambdoidal junction. Embryos were cultured in a 37°C modified whole-embryo culture system. Results: Targeted release of Wnt9b resulted in augmented Wnt expression at the lambdoidal junction. Microsurgical implantation of Wnt9b-soaked microspheres resulted in cleft correction in 27.1 percent of the Pbx-deficient embryos. The difference in the ratio of the areas of clefting between implanted and nonimplanted embryos was significant (p < 0.05). Conclusions: Ex utero correction of cleft lip with or without cleft palate in the authors’ murine model by means of microsurgical intervention and targeted delivery of Wnt proteins is an innovative and promising strategy. Although further refinement and optimization of this technique will be required to improve efficacy, the authors believe that this approach will open new avenues toward unconventional prenatal interventions for patients with cleft lip with or without cleft palate, and provide future approaches for prenatal repair of other congenital head and neck disorders.

Technological Advances in Heart and Lung Transplantation: Concomitant Cardiac Valve Surgery

This chapter covers unique features of cardiac valve surgery related to heart and lung transplantation, including pathophysiology and significance of valve disease in heart and lung transplantation, and presents a practical surgical strategy for each unique problem. We have developed a novel, simple surgical technique to prevent right ventricular failure after heart transplantation. This “commissuro-annuloplasty” procedure effectively reduces the tricuspid annular size by 2–4 cm, preventing annular dilatation and improving coaptation of the leaflets, which prevent significant tricuspid regurgitation.