Changes in cognitive ability and serum microRNA levels during aging in mice.

Mild cognitive impairment (MCI) is an early stage that can result in dementia. MCI can be reversed, and diagnosis at an early stage is crucial to control the progression to dementia. Dementia is currently diagnosed based on interviews and screening tests; however, novel biomarkers must be identified to allow early MCI detection. Therefore, the present study aimed to identify novel biomarkers in the form of blood microRNAs (miRNAs/miRs) for the diagnosis of MCI or early dementia. Blood samples were collected from C57BL/6NJcl male mice at four time points, including 4-week-old (4W), 8-week-old (8W), 36-week-old (36W) and 58-week-old (58W), and serum was isolated. Body weight and blood total cholesterol levels were increased, and blood alkaline phosphatase was decreased with aging. The 8W mice exhibited the highest cognitive ability in the Morris water maze test, whereas the 58W mice demonstrated decreased cognitive ability. The serum RNA concentrations of the 4W, 8W, 36W and 58W mice demonstrated no significant differences. Furthermore, small RNA levels were detected in the serum of all mice. miRNA microarray analysis revealed a >1.5-fold increase in the serum expression of two miRNAs (miR-21a-5p and miR-92a-3p) and a >1.5-fold decrease in the serum expression of two other miRNAs (miR-6769b-5p and miR-709) in 58W mice compared with those in 8W mice. In the future, we aim to further analyze aged mice to discover novel MCI biomarkers.

Development of Hydrophobic Cell-Penetrating Stapled Peptides as Drug Carriers.

Cell-penetrating peptides (CPPs) are widely used for the intracellular delivery of a variety of cargo molecules, including small molecules, peptides, nucleic acids, and proteins. Many cationic and amphiphilic CPPs have been developed; however, there have been few reports regarding hydrophobic CPPs. Herein, we have developed stapled hydrophobic CPPs based on the hydrophobic CPP, TP10, by introducing an aliphatic carbon side chain on the hydrophobic face of TP10. This side chain maintained the hydrophobicity of TP10 and enhanced the helicity and cell penetrating efficiency. We evaluated the preferred secondary structures, and the ability to deliver 5(6)-carboxyfluorescein (CF) as a model small molecule and plasmid DNA (pDNA) as a model nucleotide. The stapled peptide F-3 with CF, in which the stapling structure was introduced at Gly residues, formed a stable α-helical structure and the highest cell-membrane permeability via an endocytosis process. Meanwhile, peptide F-4 demonstrated remarkable stability when forming a complex with pDNA, making it the optimal choice for the efficient intracellular delivery of pDNA. The results showed that stapled hydrophobic CPPs were able to deliver small molecules and pDNA into cells, and that different stapling positions in hydrophobic CPPs can control the efficiency of the cargo delivery.

In silico optimization of peptides that inhibit Wnt/ß-catenin signaling.

The Wnt/ß-catenin signaling pathway causes transcriptional activation through the interaction between ß-catenin and T cell-specific transcription factor (TCF) and regulates a wide variety of cellular responses, including proliferation, differentiation and cell motility. Excessive transcriptional activation of the Wnt/ß-catenin pathway is implicated in developing or exacerbating various cancers. We have recently reported that liver receptor homolog-1 (LRH-1)-derived peptides inhibit the ß-catenin/TCF interaction. In addition, we developed a cell-penetrating peptide (CPP)-conjugated LRH-1-derived peptide that inhibits the growth of colon cancer cells and specifically inhibits the Wnt/ß-catenin pathway. Nonetheless, the inhibitory activity of the CPP-conjugated LRH-1-derived peptide was unsatisfactory (ca. 20 µM), and improving the bioactivity of peptide inhibitors is required for their in vivo applications. In this study, we optimized the LRH-1-derived peptide using in silico design to enhance its activity further. The newly designed peptides showed binding affinity toward ß-catenin comparable to the parent peptide. In addition, the CPP-conjugated stapled peptide, Penetratin-st6, showed excellent inhibition (ca. 5 µM). Thus, the combination of in silico design by MOE and MD calculations has revealed that logical molecular design of PPI inhibitory peptides targeting ß-catenin is possible. This method can be also applied to the rational design of peptide-based inhibitors targeting other proteins.

Development of a penetratin-conjugated stapled peptide that inhibits Wnt/ß-catenin signaling.

Wnt/ß-catenin pathway triggers the formation of a complex between ß-catenin and T cell-specific transcription factor (TCF), which induces transcriptional activation. Excessive transcriptional activation of this pathway is associated with the development, cause, and deterioration of various cancers. Therefore, the Wnt/ß-catenin pathway is an attractive drug target for cancer therapeutics and small molecule- and peptide-based protein-protein interaction (PPI) inhibitors have been developed. However, peptide-based PPI inhibitors generally have low cell-membrane permeability because of their large molecular size. To improve cell-membrane permeability, conjugating cell-penetrating peptides (CPPs) to PPI-inhibiting peptides is a useful method for developing intracellularly targeted PPI inhibitors. In this study, we focused on the interaction between ß-catenin and liver receptor homologue-1 (LRH-1) and designed and synthesized a series of LRH-1-derived peptides to develop inhibitors against Wnt/ß-catenin signaling. The results showed that a penetratin-conjugated LRH-1-derived peptide (Penetratin-st7) predominantly inhibited DLD-1 cell growth at 20 µM treatment via inhibition of the Wnt signaling pathway. This result suggests that Penetratin-st7 is one of promising PPI inhibitors between TCF and ß-catenin.

Posterior auricular artery free flap reconstruction of the retroauricular sulcus in microtia repair.

BACKGROUND: Autologous repair using costal cartilage grafts remains the most widely accepted method of microtia reconstruction. A major complication of current techniques is loss of ear shape caused by scarring, contracture and cartilage absorption. We present a new surgical technique utilizing the posterior auricular artery free flap in microsurgical reconstruction of the retroauricular sulcus in microtia. METHOD: Reconstruction is performed in two stages. In the first stage, a fabricated costal cartilage framework is inserted into a skin pocket as described by Nagata. In the second stage, the ear framework is elevated from the scalp and held by an additional cartilage wedge. Following indocyanine green angiography perforator mapping, a posterior auricular artery perforator flap is harvested from the contralateral (normal) ear and used to reconstruct the posterior auricular sulcus covering the cartilage framework and elevating wedge. RESULTS: The technique was applied to three patients aged 11-15 years with a follow-up time of 8 months to 3 years. The average flap artery diameter was 0.73 mm and the vein was 0.7 mm. Venous congestion occurred in one case and was resolved with a vein graft leading to complete flap recovery. Good ear shape, elevation, projection, skin color and texture were achieved in all the cases. CONCLUSION: Posterior auricular artery flap reconstruction of the retroauricular sulcus in microtia repair is a useful alternative to the current skin graft and tissue expander-based techniques. It provides the ideal skin color and texture match and may improve the overall results of microtia reconstruction by enhancing vascularity.

Challenges in global reconstructive microsurgery: The sub-Saharan african surgeons' perspective.

BACKGROUND: Microsurgery is an essential element of plastic surgery practice. However, it remains unavailable or rudimentary in several developing countries, especially in sub-Saharan Africa. This study presents the local plastic surgeons experience, while focusing on specific challenges encountered and methods to improve the sub-Saharan global microsurgery practice. METHODOLOGY: An online survey was sent to all plastic surgeons registered with the College of Surgeons East Central and Southern Africa and respective national plastic surgical societies in the east central and southern Africa regional community. A total of 57 questionnaires were sent. Surgeons' country of practice, years of experience and rate of performing microsurgical procedures were considered. RESULTS: The survey response rate was 56% (n = 32). Most participants believed microsurgery was essential in the region. The leading challenge was inadequate perioperative care, mainly attributed to shortage of support staff (n = 29, 91%). Others were lack of surgical expertise and resources. Interestingly, public unawareness of the benefits of microsurgery was also noted as a critical hindrance. The foremost suggestion on improvement (n = 19, 59%) was to enhance training with a multidisciplinary team-building approach. Others included increased advocacy, publications and funding. CONCLUSION: The Plastic surgeons' perspective recognizes the needs of Global Reconstructive Microsurgery in sub-Saharan Africa. However, inadequate perioperative care, insufficient expertise, lack of equipment and lack of public awareness were major hindrances. Finally, there is a need to improve microsurgery in the region through advocacy, training and multidisciplinary team building.

Local Flaps with Negative Pressure Wound Therapy in Secondary Reconstruction of Myelomeningocele Wound Necrosis.

Major wound necrosis is an uncommon yet critical complication of meningomyelocele surgical repair with few reports available. Management is demanding and often requires further reconstructive surgery. We report a case of a neonate who developed extensive wound necrosis with dehiscence following primary repair of myelomeningocele. The large defect was reconstructed using transposition fasciocutaneous flaps and negative pressure wound therapy applied over the flap donor sites resulting in wound closure, alleviating the need for further surgery. We report this case to highlight the importance of local flap design in reconstruction of the complex wounds seen following meningomyelocele repair wound necrosis. Additionally, we report the unique utilization of negative pressure wound therapy in the management of myelomeningocele.