Reconstruction of a Severe Open Tibiofibular Fracture using an Ipsilateral Vascularized Fractured Fibula with a Thoracodorsal Artery Perforator Free Flap.

The Gustilo IIIB tibiofibular fractures often result in long bone loss and extensive soft tissue defects. Reconstruction of these complex wounds is very challenging, especially when it includes long bone grafts, because the donor site is limited. We describe our experience using a set of chimeric ipsilateral vascularized fibula grafts with a thoracodorsal artery perforator free flap to reconstruct the traumatic tibia defects. A 66-year-old male suffered a severe comminuted tibia fracture and segmented fibula fracture with large soft tissue defects as a result of a traffic accident. He also had an open calcaneal fracture with soft tissue defects on the ipsilateral side. All the main vessels of the lower extremity were intact, and the cortical bone defect of the tibia was almost as large as the fractured fibula segment. We used an ipsilateral vascularized fibula graft to reconstruct the tibia and a thoracodorsal artery perforator flap to resurface the soft tissue, using the distal ends of peroneal vessels as named into sequential chimeric flaps. After 3 weeks, the calcaneal defect was reconstructed with second thoracodorsal artery perforator free flap. Reconstruction was successful and allowed rapid rehabilitation because of reduced donor site morbidity.

A modified induced membrane 2-stage technique using a thoracodorsal artery perforator free flap followed by vascularized or non-vascularized free fibular transfer for the treatment of complex bone infection with concomitant severe soft tissue lesion-A case series of 9 cases.

Treatment of post-traumatic complex bone infection is very challenging. The two principal bone reconstruction approaches are the single-stage vascularized bone graft technique and the two-stage induced membrane technique (IMT). Here we introduce a modified 2-stage induced membrane technique (MIMT) for complex long bone infection with a major bone defect and a concomitant severe soft tissue lesion. The 2-stage procedure consists of bone debridement, placement of a PMMA spacer and soft tissue reconstruction with a thoracodorsal artery perforator free flap ("Tdap") at stage 1. At stage 2, the thoracodorsal artery perforator flap is elevated and a fibular strut graft (either vascularized of non-vascularized) is placed for bone reconstruction. We retrospectively analyzed the extents of lower extremity, long bone, post-traumatic bone infection treated via MIMT from 2008 to 2020. There were nine such cases (eight males) of mean age 59.8 (range 31 to 79) years. The osteomyelitis durations ranged from 3 to 360 months (mean 53 months). The cortical bone defect sizes was ranged from 9 to 14 cm (mean10.7 cm). All skin resurfacing employed Tdap. Vascularized fibular grafts were placed in six patients and non-vascularized grafts were placed in three. The fibular graft size ranged from 12.5 to 19 cm (mean 16.2 cm). Non-vascularized iliac bone grafts served as the fibula docking sites. Unfortunately, all patients suffered complications before bone union was achieved. One case of plate stress fracture and one case of screw fracture required plate and screw change. In three cases of cellulitis, one resolved by use of intravenous antibiotics, others required plate and screw removal. Wound disruption required re-suture and distal skin flap partial necrosis was covered by perforator-based island flap. One case of fibular stress fracture needed cast for 4 weeks. A peroneal nerve palsy patient recovered spontaneously. Bone union was achieved after 6 months in five patients and after 8 months in three (mean 6.9 months). All patients were able to walk unaided. The follow-up period ranged from 2 to 14 years (mean 6.2 years). MIMT saves the limbs in cases with difficult post-traumatic bone infection. It is valid treatment option for complex bone infections with severe soft tissue lesions. However, even with this technique potential complication must be considered.

Fingertip coverage with uni-pedicled volar rotational advancement flap with large Z-plasty: a report on 112 cases.

BACKGROUND: Simple and safe fingertip reconstruction methods involve the use of local neurovascular islands flaps that can preserve functional length and sensitivity, and reconstruction with skin of the same texture. However, techniques involving flaps have numerous drawbacks and do not satisfy all the requirements for fingertip reconstruction. A particular problem is the persistence of contracture deformity due to lack of full flap advancement. We present a new technique using uni-pedicled volar rotational advancement flap with large Z-plasty, and describe the results of long-term follow-up. METHODS: From October 1993 to December 2009, 112 fingers of 98 patients were covered with uni-pedicled volar rotational advancement flap with large Z-plasty after sustaining various types of injuries or finger pulp avulsion. A longitudinal incision was made along the lateral border of the digit and a large neurovascular volar flap was elevated just above the pulleys and flexor tendon sheath. To release tension, a large Z-plasty was applied at the metacarpophalangeal joint or interphalangeal joint crease. The final patient outcomes were reviewed retrospectively. RESULTS: All fingertip injuries were treated without flap necrosis. Partial wound dehiscence was observed in two patients and average static two-point discrimination was 5.2 mm. There were no postoperative contracture deformities, joint stiffness, paresthesia, or hypersensitivity. Most patients were left with acceptable scarring and were free of postoperative pain and cold intolerance during the long-term follow-up. CONCLUSIONS: Our novel technique provides durable, completely sensate, and well-vascularized coverage of the fingertip with minimal discomfort to patients.

Rad51 Regulates Reprogramming Efficiency through DNA Repair Pathway.

Rad51 is a key component of homologous recombination (HR) to repair DNA double-strand breaks and it forms Rad51 recombinase filaments of broken single-stranded DNA to promote HR. In addition to its role in DNA repair and cell cycle progression, Rad51 contributes to the reprogramming process during the generation of induced pluripotent stem cells. In light of this, we performed reprogramming experiments to examine the effect of co-expression of Rad51 and four reprogramming factors, Oct4, Sox2, Klf4, and c-Myc, on the reprogramming efficiency. Co-expression of Rad51 significantly increased the numbers of alkaline phosphatase-positive colonies and embryonic stem cell-like colonies during the process of reprogramming. Co-expression ofRad51 significantly increased the expression of epithelial markers at an early stage of reprogramming compared with control cells. Phosphorylated histone H2AX (γH2AX), which initiates the DNA double-strand break repair system, was highly accumulated in reprogramming intermediates upon co-expression of Rad51. This study identified a novel role of Rad51 in enhancing the reprogramming efficiency, possibly by facilitating mesenchymal-to-epithelial transition and by regulating a DNA damage repair pathway during the early phase of the reprogramming process.

Novel Function of Lysine Methyltransferase G9a in the Regulation of Sox2 Protein Stability.

G9a is a lysine methyltransferase (KMTase) for histone H3 lysine 9 that plays critical roles in a number of biological processes. Emerging evidence suggests that aberrant expression of G9a contributes to tumor metastasis and maintenance of a malignant phenotype in cancer by inducing epigenetic silencing of tumor suppressor genes. Here, we show that G9a regulates Sox2 protein stability in breast cancer cells. When G9a lysine methyltransferase activity was chemically inhibited in the ER(+) breast cancer cell line MCF7, Sox2 protein levels were decreased. In addition, ectopic overexpression of G9a induced accumulation of Sox2. Changes in cell migration, invasion, and mammosphere formation by MCF7 cells were correlated with the activity or expression level of G9a. Ectopic expression of G9a also increased Sox2 protein levels in another ER(+) breast cancer cell line, ZR-75-1, whereas it did not affect Sox2 expression in MDA-MB-231 cells, an ER(-) breast cancer cell line, or in glioblastoma cell lines. Furthermore, treatment of mouse embryonic stem cells with a KMT inhibitor, BIX-01294, resulted in a rapid reduction in Sox2 protein expression despite increased Sox2 transcript levels. This finding suggests that G9a has a novel function in the regulation of Sox2 protein stability in a cell type-dependent manner.

Rad51 regulates cell cycle progression by preserving G2/M transition in mouse embryonic stem cells.

Homologous recombination (HR) maintains genomic integrity against DNA replication stress and deleterious lesions, such as double-strand breaks (DSBs). Rad51 recombinase is critical for HR events that mediate the exchange of genetic information between parental chromosomes in eukaryotes. Additionally, Rad51 and HR accessory factors may facilitate replication fork progression by preventing replication fork collapse and repair DSBs that spontaneously arise during the normal cell cycle. In this study, we demonstrated a novel role for Rad51 during the cell cycle in mouse embryonic stem cells (mESCs). In mESCs, Rad51 was constitutively expressed throughout the cell cycle, and the formation of Rad51 foci increased as the cells entered S phase. Suppression of Rad51 expression caused cells to accumulate at G2/M phase and activated the DNA damage checkpoint, but it did not affect the self-renewal or differentiation capacity of mESCs. Even though Rad51 suppression significantly inhibited the proliferation rate of mESCs, Rad51 suppression did not affect the replication fork progression and speed, indicating that Rad51 repaired DNA damage and promoted DNA replication in S phase through an independent mechanism. In conclusion, Rad51 may contribute to G2/M transition in mESCs, while preserving genomic integrity in global organization of DNA replication fork.

Lefty1 and lefty2 control the balance between self-renewal and pluripotent differentiation of mouse embryonic stem cells.

Lefty expression has been recognized as a stemness marker because Lefty is enriched both in undifferentiated embryonic stem cells (ESCs) and in blastocysts. Here, we examined the function of Lefty1 and Lefty2 in the maintenance of self-renewal and pluripotency of mouse ESCs (mESCs). Suppression of Lefty1 or Lefty2 expression in mESCs did not alter the self-renewal properties of mESCs under nondifferentiating conditions, but suppression of these genes did affect Smad2 phosphorylation and differentiation. Lefty1 knockdown mESCs showed enhanced phosphorylation of Smad2 and increased differentiation potential, whereas Lefty2 knockdown mESCs exhibited reduced phosphorylation of Smad2 and enhanced self-renewal in the presence of a differentiation signal. In vivo, teratomas developed from Lefty2 knockdown mESCs contained massive expansions of immature neuroepithelium, a marker of malignant teratomas. Taken together, these results suggest that optimal expression of Lefty1 and Lefty2 is critical for the balanced differentiation of mESCs into three germ layers.

TCEA3 binds to TGF-beta receptor I and induces Smad-independent, JNK-dependent apoptosis in ovarian cancer cells.

TFIIS is a transcription elongation factor conserved in frog, mouse and human. Recently, knockdown of TCEA1, the most well-characterized isoform of TFIIS, by RNA silencing was reported to inhibit cancer cell proliferation and induce apoptosis in breast, lung and pancreatic cancer cell lines through activation of p53 (Hubbard et al., 2008 [1]). However, the functions of other TFIIS isoforms are poorly defined. The present study shows that TCEA3, an isoform of TFIIS, can trigger ovarian cancer-specific cell death by activating the JNK signaling pathway. TCEA3 expression is low in ovarian cancer cell lines compared to noncancerous ovarian epithelial cells. Suppression of TCEA3 in noncancerous ovarian epithelial cells promotes cell growth whereas ectopic expression of TCEA3 in ovarian cancer cell lines induces the caspase-dependent mitochondrial cell death pathway. Molecular and chemical inhibition assays show that the interaction of TCEA3 with TGFß receptor I induces cell death in ovarian cancer cell through Smad-independent activation of the JNK pathway. These results reveal that TCEA3 induces a novel apoptotic mechanism in OEC, which provides TCEA3 as a novel target to develop therapeutics of ovarian cancer.

PI3K/Akt and Stat3 signaling regulated by PTEN control of the cancer stem cell population, proliferation and senescence in a glioblastoma cell line.

Malignant gliomas are the most common primary brain tumor in adults. A number of genes have been implicated in glioblastoma including mutation and deletion of PTEN. PTEN is a regulator of PI3K-mediated Akt signaling pathways and has been recognized as a therapeutic target in glioblastoma. To achieve potent therapeutic inhibition of the PI3K-Akt pathway in glioblastoma, it is essential to understand the interplay between the regulators of its activation. Here, ectopic expression of PTEN in the U-87MG human glioblastoma-astrocytoma cell line is shown to result in the depletion of glioblastoma stem cells (GSCs) and to cause growth retardation and senescence. These effects are likely to be associated with PTEN-mediated cooperative perturbation of Akt and Stat3 signals. Using an in vivo rat model of glioblastoma, we showed that PTEN-overexpressing U-87MG cells failed to induce tumor formation, while untreated U-87MG cells did so. Furthermore, cells expressing the phosphorylated form of Stat3 were completely absent from the brain of rats implanted with PTEN-overexpressing U-87MG cells. Based on these results, PTEN appears to function as a crucial inhibitor of GSCs and as an inducer of senescence, suggesting that functional enhancement of the PTEN pathway will be useful to provide a therapeutic strategy for targeting glioblastoma.

Sprouty1 regulates neural and endothelial differentiation of mouse embryonic stem cells.

Fibroblast growth factor (FGF) signaling is implicated in the control of pluripotency and lineage differentiation of both human and mouse embryonic stem cells (mESCs). FGF4 dependent stimulation of ERK1/2 signaling triggers transition of pluripotent ESCs from self-renewal and lineage commitment. In this study, Sprouty 1 (Spry1) expression was observed in undifferentiated mESCs, where it modulated ERK1/2 activity. Spry1 was confirmed as dispensable for the maintenance of self-renewal. However, suppression of Spry1 expression and subsequent activation of ERK1/2 signaling promoted neural differentiation and inhibited endothelial differentiation of mESCs. Moreover, evidence is presented which indicates that SHP2, a major determinant of balance between mESC self-renewal and differentiation, directly regulates Spry1 activity to modulate ERK1/2 signaling and lineage-specific differentiation in mESCs. Our results show that Spry1 has an essential role in the lineage specific differentiation of mESCs.