Cellular protection from H2O2 toxicity by Fv-Hsp70: protection via catalase and gamma-glutamyl-cysteine synthase.

Heat shock proteins (HSPs), especially Hsp70 (HSPA1), have been associated with cellular protection from various cellular stresses including heat, hypoxia-ischemia, neurodegeneration, toxins, and trauma. Endogenous HSPs are often synthesized in direct response to these stresses but in many situations are inadequate in protecting cells. The present study addresses the transduction of Hsp70 into cells providing protection from acute oxidative stress by H2O2. The recombinant Fv-Hsp70 protein and two mutant Fv-Hsp70 proteins minus the ATPase domain and minus the ATPase and terminal lid domains were tested at 0.5 and 1.0 µM concentrations after two different concentrations of H2O2 treatment. All three recombinant proteins protected SH-SY5Y cells from acute H2O2 toxicity. This data indicated that the protein binding domain was responsible for cellular protection. In addition, experiments pretreating cells with inhibitors of antioxidant proteins catalase and gamma-glutamylcysteine synthase (GGCS) before H2O2 resulted in cell death despite treatment with Fv-Hsp70, implying that both enzymes were protected from acute oxidative stress after treatment with Fv-Hsp70. This study demonstrates that Fv-Hsp70 is protective in our experiments primarily by the protein-binding domain. The Hsp70 terminal lid domain was also not necessary for protection.

Effects of Priming Discriminated Experiences on Emotion Recognition Among Asian Americans.

This study explored the priming effects of discriminated experiences on emotion recognition accuracy of Asian Americans. We hypothesized that when Asian Americans were reminded of discriminated experiences due to their race, they would detect subtle negative emotional expressions on White faces more accurately than would Asian Americans who were primed with a neutral topic. This priming effect was not expected to emerge in detecting negative facial expressions on Asian faces. To test this hypothesis, 108 participants were randomly assigned to one of two conditions: write about their experiences with racial discrimination (experimental) or write about a neutral topic (control). Then, they were given an emotion recognition test consisting of White and Asian faces. The current study found a significant interaction effect of priming condition by target race. When Asian Americans were reminded of discriminated experiences, they displayed heightened sensitivity to negative emotional expressions on White faces, but not to the negative expressions on Asian faces. The implications of these findings were discussed.

Efficient endothelial and smooth muscle cell differentiation from human pluripotent stem cells through a simplified insulin-free culture system.

A major obstacle for using human pluripotent stem cells (hPSCs) derived vascular cells for cell therapy is the lack of simple, cost-saving, and scalable methods for cell production. Here we described a simplified and chemically defined medium (AATS) for endothelial cells (ECs) and smooth muscle cells (SMCs) differentiation. AATS medium does not contain insulin, enabling the rapid and highly efficient vascular mesoderm formation through accelerating metabolic and autophagy-enhanced mesoderm induction. Transcriptome profiling confirmed that hPSC-derived ECs and SMCs in the AATS medium closely resembled primary ECs and SMCs formed in vivo. ECs appeared to adhere and grow better in the AATS medium over other cell types, which allowed the purification of CD31+CD144+ double-positive cells. Furthermore, the AATS medium was compatible with 3D microscaffold (MS) culture, which may facilitate large-scale bioproduction of ECs. HPSC-derived ECs and SMCs in the AATS medium exhibited strong revascularization potential in treating murine ischemic models. Our study provided a cost-effective and efficient medium system to manufacture GMP compatible, off-the-shelf ECs, and SMCs to model human diseases and vascular repair.

Biphasic modulation of insulin signaling enables highly efficient hematopoietic differentiation from human pluripotent stem cells.

BACKGROUND: Hematopoietic lineage cells derived from human pluripotent stem cells (hPSCs) hold great promise for the treatment of hematological diseases and providing sufficient cells for immune therapy. However, a simple, cost-effective method to generate large quantities of hematopoietic stem/progenitor cells (HSPCs) is not yet available. METHODS: We established a monolayer, chemically defined culture system to induce hematopoietic differentiation from hPSCs in 8 days. RESULTS: We found that insulin-free medium allowed hPSCs to leave pluripotency promptly and preferably enter the vascular lineage. Addition of insulin during the later stage of differentiation was essential for the efficient induction of hemogenic endothelium and the emergence of large numbers of CD34+CD43+ HSPCs, while no insulin condition preferably permits endothelial differentiation. Global transcriptome profiling revealed that HSPCs differentiated using our protocol were similar to embryoid body-derived HSPCs. HSPCs obtained from our differentiation system formed robust erythroid, granulocyte and monocyte/macrophage colonies in CFU assay, and can be induced to generate functional macrophages with robust phagocytic ability. CONCLUSION: Our results demonstrated that proper manipulation of insulin-mTOR signaling can greatly facilitate HSPC formation. This finding can be further exploited to formulate cost-effective differentiation medium to generate large quantities of cells of desired blood lineages for regenerative medicine.

Use of the Ipsilateral Anteromedial Thigh Flap for Immediate Rescue of Nonviable Anterolateral Thigh Flaps in Head and Neck Cancer Reconstruction.

BACKGROUND: It is not always possible to use the anatomically variable free anterolateral thigh (ALT) flap for reconstructive surgery. An anteromedial thigh (AMT) flap serves as a good alternative, and shares the same vascular pedicle as the ALT flap. METHODS: Of 698 reconstructions performed in 2006 to 2013 following head and neck tumor ablation surgery, ALT flaps were used in 653 patients. Eighteen free AMT flaps were harvested to replace variant nonviable ALT flaps. RESULTS: The lack of a sizable perforator in the ALT flap territory was the main reason for changing the reconstruction plan. Anteromedial thigh flap size ranged from 10 × 4 to 30 × 8 cm. The flap survival rate was 100%. The follow-up period ranged from 3 to 56 months. CONCLUSION: During head and neck reconstruction, when no sizable perforator is available during harvest of the ALT flap, successful reconstruction can be achieved using the ipsilateral AMT flap without additional donor-site morbidity.

Optimizing mesoderm progenitor selection and three-dimensional microniche culture allows highly efficient endothelial differentiation and ischemic tissue repair from human pluripotent stem cells.

BACKGROUND: Generation of large quantities of endothelial cells is highly desirable for vascular research, for the treatment of ischemia diseases, and for tissue regeneration. To achieve this goal, we developed a simple, chemically defined culture system to efficiently and rapidly differentiate endothelial cells from human pluripotent stem cells by going through an MESP1 mesoderm progenitor stage. METHODS: Mesp1 is a key transcription factor that regulates the development of early cardiovascular tissue. Using an MESP1-mTomato knock-in reporter human embryonic stem cell line, we compared the gene expression profiles of MESP1+ and MESP1- cells and identified new signaling pathways that may promote endothelial differentiation. We also used a 3D scaffold to mimic the in vivo microenvironment to further improve the efficiency of endothelial cell generation. Finally, we performed cell transplantation into a critical limb ischemia mouse model to test the repairing potential of endothelial-primed MESP1+ cells. RESULTS: MESP1+ mesoderm progenitors, but not MESP1- cells, have strong endothelial differentiation potential. Global gene expression analysis revealed that transcription factors essential for early endothelial differentiation were enriched in MESP1+ cells. Interestingly, MESP1 cells highly expressed Sphingosine-1-phosphate (S1P) receptor and the addition of S1P significantly increased the endothelial differentiation efficiency. Upon seeding in a novel 3D microniche and priming with VEGF and bFGF, MESP1+ cells markedly upregulated genes related to vessel development and regeneration. 3D microniches also enabled long-term endothelial differentiation and proliferation from MESP1+ cells with minimal medium supplements. Finally, we showed that transplanting a small number of endothelial-primed MESP1+ cells in 3D microniches was sufficient to mediate rapid repair of a mouse model of critical limb ischemia. CONCLUSIONS: Our study demonstrates that combining MESP1+ mesoderm progenitor cells with tissue-engineered 3D microniche and a chemically defined endothelial induction medium is a promising route to maximizing the production of endothelial cells in vitro and augment their regenerative power in vivo.

Functional Outcome Analysis After Anterolateral Thigh Flap Reconstruction of Pharyngoesophageal Defect.

BACKGROUND: Pharyngoesophageal defects are traditionally reconstructed with jejunal or radial forearm flaps. Recently, anterolateral thigh flaps have served for pharyngoesophageal reconstruction. We tell of our experience with anterolateral thigh flap for the reconstruction of pharyngoesophageal defect for the past 5 years. This study presents another modified flap design and refinement of surgical techniques. METHODS: In all, 45 pharyngoesophageal reconstructions were performed during 2006 to 2011, namely patch (n = 18), near-circumferential (n = 8), and circumferential defect (n = 19) reconstructed by 2 plastic surgeons with the same trapezoid anterolateral thigh flap design. RESULTS: Total flap loss occurred in 2 patients (4%). Two patients experienced partial flap necrosis. Fistulas occurred in 8 patients, but only 5 of these needed further suture ligation or local flap treatment. Postoperative strictures without tumor recurrence were identified in 4 patients (9%). Forty patients (88.9%) could tolerate oral diet; 34 of these ate soft or solid food. CONCLUSIONS: Anterolateral thigh flap offers good coverage and swallowing function in the reconstruction of hypopharyngeal defect. Trapezoid drawing of anterolateral thigh flap makes the design simplified, with an accepted complication rate.

Restoration of glans penis defect by a bipedicled foreskin flap.

Plastic surgeons reconstruct hard and soft tissues in many parts of the human body. Penile reconstruction is unique in that the target tissue has to be soft but intermittently rigid. There are many ways to treat penile defects. The ideal reconstruction must take into considerations sensation, cosmesis, and erectile functions. There is limited literature available on the management of penile glans defect. In this report, we present the reconstruction of penile glans defect, after surgical excision of Bowen disease, using a bipedicled foreskin flap of bucket handle type. It is easy to perform and highly effective, and importantly, both cosmetic and functional outcomes at 1-year follow-up were quite satisfactory.

Reflections on a decade of funding public hospital systems.

As the nation embarks upon health reform, many questions remain unanswered. Important among them is the fate of public hospitals, which have historically cared for the uninsured. Under health reform, public hospitals will face marketplace competition to serve newly insured people. Can public hospitals change, so that they can survive and thrive in a competitive environment? This article describes lessons learned from a decade of funding by the California HealthCare Foundation to improve clinical care in California's public hospitals. It also identifies factors that will influence California's public hospitals in the coming months and years.

Management of infected groin wounds after vascular surgery.

BACKGROUND: Management of an infected groin wound after vascular surgery may be a challenge. We report a retrospective series of cases of the management of groin defects and an algorithm for their management based on our own experience and related literature. PATIENTS AND METHODS: We conducted a retrospective chart review from June 2008 to February 2012 of patients with infected groin wounds after vascular surgical procedures. The review disclosed six patients with a history of femoral cannulation or extracorporeal membrane oxygenation (ECMO), one patient with a femorofemoral bypass, one patient with an intra-aortic balloon pump (IABP), and one patient with a thoracoabdominal aneurysm following stent implantation. Exposure of femoral vessels was noted in seven of these nine patients, and wound cultures of all nine patients yielded positive results. RESULTS: The mean age of the nine patients (five males and four females) was 54.6 years (range 17-79 years). The mean follow-up was 13.6 months (range 8-30 months). Four of the patients were treated with a pedicled gracilis flap; one with a local flap; one with a myocutaneous flap of the anterolateral thigh (ALT) combined with a partial tensor fascia lata (TFL) flap; one with primary closure; and two with a myocutaneous island pedicle flap of the ALT. No donor-site complications were noted. There was partial skin cyanosis in the patient treated with a myocutaneous flap of the ALT combined with a TFL flap, which resolved after one week. The scheduled follow-up of the patients showed that all of their groin wounds had healed well. CONCLUSIONS: A pedicled flap of gracilis muscle is an ideal and effective option for covering infected groin wounds of <10 cm with exposure of femoral vessels. According to a literature review, a sartorius muscle flap is another option for accomplishing this. A myocutaneous island flap of the ALT is indicated for infected groin wounds>10 cm with exposure of femoral vessels. The literature indicates that myocutaneous flaps of rectus abdominis (RA) muscle and flaps of rectus femoris (RF) muscle are also suitable for groin wounds larger than 10 cm. Bilateral flaps of ALT and bilateral myocutaneous flaps of RA or RF muscle are suggested for the reconstruction of bilateral groin wounds. For infected groin wounds without exposure of femoral vessels, a local flap or primary closure are suggested, depending on the size of the defect.

Engineered autologous bone marrow mesenchymal stem cells: alternative to cleft alveolar bone graft surgery.

Human recombinant bone morphogenetic protein 2 (rhBMP-2) accelerates bone regeneration but is associated with limited cementum and periodontal ligament regeneration, local root resorption, and ankylosis. This study assessed a new approach to the regeneration of the alveolar bone and periodontal attachment apparatus using a combination of ex vivo autologous bone marrow mesenchymal stem cells (MSCs) engineered by replication defective adenovirus to express the BMP-2 gene and pluronic F127 (PF127) in a large mammalian animal model. Bilateral maxillary periodontal defects were created over the premolar area in 9 mature male miniature swine. The 18 defects were randomly assigned to receive either BMP-2-expressing MSCs in the advBMP-2 group or MSCs alone in the MSC group. The regenerated periodontal attachment apparatus was evaluated histologically, and the total regenerated bone volume was calculated from three-dimensional computed tomography analysis. Three months after implantation, significant bone volume was regenerated in the advBMP-2 group. Periodontal apparatus regeneration was significantly better in the advBMP-2 group. New cementum and Sharpey fibers were observed on the denuded root surfaces in the advBMP-2 group, whereas incomplete healing with localized root surface resorption was noted in the control group. The use of ex vivo BMP-2-engineered autologous MSCs enhanced bone and periodontal apparatus regeneration in maxillary alveolar and periodontal defects in swine. This novel integrated approach might be suitable for clinical periodontal apparatus repair. This may be an alternative for cleft alveolar bone graft surgery.

Distinctive genital folliculosebaceous cystic hamartomas in a patient with fibrous dysplasia.

Folliculosebaceous cystic hamartoma is a benign tumor composed of follicular, sebaceous, and mesenchymal components. Usually, it appears in adulthood as a dome or a pedunculated nodule on the face, especially the nose. Extracephalic lesions are sporadically reported, such as on the upper back, forearm, ear, labia majora, and the nipple. Herein, we report a patient with left maxillary fibrous dysplasia who experienced bilateral labia majora disfigurement after her puberty. She experienced physical distress because of tenderness due to the tangled, stretched hamartomas and psychological distress because of failure to have sexual contacts. The exophytic dark skin-colored fusing polypoid labia majora deformities were excised, and the folliculosebaceous cystic hamartoma was impressed by the pathology. We used an advancing flap for labiaplasty, and the outcome was aesthetically satisfactory. There is no recurrence in the 6-month follow-up period. To our knowledge, this is the first report of a folliculosebaceous cystic hamartoma on the bilateral labia majora in a patient with fibrous dysplasia. This extraordinary presentation is much different from others owing to its unusual appearance and a larger size.