Improvement of survival of skin flaps by combined gene transfer of hepatocyte growth factor and prostacyclin synthase

BACKGROUND: Increasing the local blood flow is a critical factor for long-term survival of skin flaps. Thus, a molecular therapy to increase the blood flow by means of an angiogenic factor is considered to be a useful strategy to improve skin flap survival. We focused on a combined strategy to stimulate not only angiogenesis, but also vasodilation of local microvessels, using co-transfection of the hepatocyte growth factor (HGF) and prostacyclin synthase (PGIS) genes to enhance the survival of random-pattern skin flaps. METHODS AND RESULTS: A 2 x 8 cm full thickness cranial pedicled random-pattern flap was made on the back of each 12-week-old male rat. At 3 days before operation, 400 microg of human HGF and PGIS naked plasmid DNA or control plasmid was transfected into the flaps by needle-less injection using a Shima Jet, resulting in successful expression of human HGF and PGIS in the skin flaps. Transfection of both genes into the distal half of skin flaps at 3 days prior to operation significantly increased the survival rate of skin flaps, while transfection all over the flaps did not. In addition, transfection prior to operation was more effective than simultaneous treatment. Moreover, co-transfection of these genes improved the survival area of skin flaps, accompanied by an increase in blood flow of skin flaps, even in a diabetic model. CONCLUSIONS: Overall, these results indicate that combination treatment with HGF and PGIS genes by Shima Jet could be an effective strategy to improve skin flap survival.     

Structural basis for recruitment of human flap endonuclease 1 to PCNA

Flap endonuclease-1 (FEN1) is a key enzyme for maintaining genomic stability and replication. Proliferating cell nuclear antigen (PCNA) binds FEN1 and stimulates its endonuclease activity. The structural basis of the FEN1-PCNA interaction was revealed by the crystal structure of the complex between human FEN1 and PCNA. The main interface involves the C-terminal tail of FEN1, which forms two beta-strands connected by a short helix, the betaA-alphaA-betaB motif, participating in beta-beta and hydrophobic interactions with PCNA. These interactions are similar to those previously observed for the p21CIP1/WAF1 peptide. However, this structure involving the full-length enzyme has revealed additional interfaces that are involved in the core domain. The interactions at the interfaces maintain the enzyme in an inactive 'locked-down' orientation and might be utilized in rapid DNA-tracking by preserving the central hole of PCNA for sliding along the DNA. A hinge region present between the core domain and the C-terminal tail of FEN1 would play a role in switching the FEN1 orientation from an inactive to an active orientation.     

Molecular docking based virtual screening of natural compounds as potential BACE1 inhibitors: 3D QSAR pharmacophore mapping and molecular dynamics analysis

Beta-site APP cleaving enzyme1 (BACE1) catalyzes the rate determining step in the generation of Aβ peptide and is widely considered as a potential therapeutic drug target for Alzheimer’s disease (AD). Active site of BACE1 contains catalytic aspartic (Asp) dyad and flap. Asp dyad cleaves the substrate amyloid precursor protein with the help of flap. Currently, there are no marketed drugs available against BACE1 and existing inhibitors are mostly pseudopeptide or synthetic derivatives. There is a need to search for a potent inhibitor with natural scaffold interacting with flap and Asp dyad. This study screens the natural database InterBioScreen, followed by three-dimensional (3D) QSAR pharmacophore modeling, mapping, in silico ADME/T predictions to find the potential BACE1 inhibitors. Further, molecular dynamics of selected inhibitors were performed to observe the dynamic structure of protein after ligand binding. All conformations and the residues of binding region were stable but the flap adopted a closed conformation after binding with the ligand. Bond oligosaccharide interacted with the flap as well as catalytic dyad via hydrogen bond throughout the simulation. This led to stabilize the flap in closed conformation and restricted the entry of substrate. Carbohydrates have been earlier used in the treatment of AD because of their low toxicity, high efficiency, good biocompatibility, and easy permeability through the blood–brain barrier. Our finding will be helpful in identify the potential leads to design novel BACE1 inhibitors for AD therapy.     

腓肠肌皮瓣联合负压封闭引流治疗胫骨慢性骨髓炎及软组织缺损的临床研究

目的:研究腓肠肌皮瓣联合负压封闭引流治疗胫骨慢性骨髓炎及软组织缺损的临床疗效。方法:选取2013年10月到2014年10月我院收治的胫骨慢性骨髓炎及软组织缺损患者68例,按照手术方式不同将患者分为研究组(n=39例)和对照组(n=29例),其中研究组给予腓肠肌皮瓣联合负压封闭引流治疗,对照组给予常规手术方法治疗,比较两组临床疗效、C反应蛋白(CRP)变化、治愈时间以及复发率。结果:研究组治愈率94.9%(37/39)显著高于对照组的75.9%(22/29),两组比较差异具有统计学意义(P0.05);研究组CRP变化和治愈时间均显著优于对照组,两组比较差异具有统计学意义(P0.05);研究组复发率显著低于对照组,两组比较差异具有统计学意义(P0.05)。结论:腓肠肌皮瓣联合负压封闭引流治疗胫骨慢性骨髓炎及软组织缺损具有较高治愈率,能显著降低炎性反应,缩短愈合时间,降低复发率。     

Interaction between APC and Fen1 during breast carcinogenesis

Aberrant DNA base excision repair (BER) contributes to malignant transformation. However, inter-individual variations in DNA repair capacity plays a key role in modifying breast cancer risk. We review here emerging evidence that two proteins involved in BER – adenomatous polyposis coli (APC) and flap endonuclease 1 (Fen1) – promote the development of breast cancer through novel mechanisms. APC and Fen1 expression and interaction is increased in breast tumors versus normal cells, APC interacts with and blocks Fen1 activity in Pol-β-directed LP-BER, and abrogation of LP-BER is linked with cigarette smoke condensate-induced transformation of normal breast epithelial cells. Carcinogens increase expression of APC and Fen1 in spontaneously immortalized human breast epithelial cells, human colon cancer cells, and mouse embryonic fibroblasts. Since APC and Fen1 are tumor suppressors, an increase in their levels could protect against carcinogenesis; however, this does not seem to be the case. Elevated Fen1 levels in breast and lung cancer cells may reflect the enhanced proliferation of cancer cells or increased DNA damage in cancer cells compared to normal cells. Inactivation of the tumor suppressor functions of APC and Fen1 is due to their interaction, which may act as a susceptibility factor for breast cancer. The increased interaction of APC and Fen1 may occur due to polypmorphic and/or mutational variation in these genes. Screening of APC and Fen1 polymorphic and/or mutational variations and APC/Fen1 interaction may permit assessment of individual DNA repair capability and the risk for breast cancer development. Such individuals might lower their breast cancer risk by reducing exposure to carcinogens. Stratifying individuals according to susceptibility would greatly assist epidemiologic studies of the impact of suspected environmental carcinogens. Additionally, a mechanistic understanding of the interaction of APC and Fen1 may provide the basis for developing new and effective targeted chemopreventive and chemotherapeutic agents.     

Soft tissue re-growth after different crown lengthening techniques among Indian patients

Patients often report complaining of fractured or decayed teeth with severe morphological deformities. However, all these clinical scenarios require the same level of care and consideration to rehabilitate form, function and esthetics. Some cases have sufficient clinical crown height while others often require an interdisciplinary approach in the form of orthodontic/surgical extrusion or surgical periodontal options. A common factor delaying treatment is soft tissue regrowth after crown lengthening which delays the impression required for final prosthesis. Therefore, it is of interest to compare the prevalence of soft tissue regrowth a week after different crown lengthening techniques including laser gingivectomy, electrocautery gingivectomy, modified Widman flap and apically repositioned. The parameters assessed included 1-week postoperative soft tissue regrowth after crown lengthening, age of patients and gender. It was observed that laser and electrocautery-assisted gingivectomy had a higher rate of soft tissue regrowth as compared to surgical techniques. It was further noted that laser and electrocautery assisted gingivectomy had a higher frequency of soft tissue rebound growth compared to surgical crown lengthening using modified widman flap and apically repositioned flap, which was statistically insignificant. Patients within the age groups of 26-60 years were found to have a higher tendency of soft tissue regrowth, which was found to be clinically and statistically significant (p<0.05).     

Mesenchymal stem cells improve survival in ischemic diabetic random skin flap via increased angiogenesis and VEGF expression

Random skin flaps (RSFs) are cutaneous flaps. Despite the negative impact of diabetes mellitus (DM) on RSF viability, they are commonly used in diabetic patients. In this study, we have assessed bone marrow mesenchymal stem cell (BMMSC) treatment on RSF survival, tensiometrical parameters, angiogenesis, and mast cells (MCs) count in an ischemic RSF model in rats with type 1 DM (T1DM). We induced T1DM in 30 Wistar adult male rats. The animals were assigned to three groups of 10 rats per group as follows: group 1 (control); group 2 (placebo), and group 3 (BMMSCs). A 30 × 80 mm RSF was created in each rat. On day 7, we measured the viable portion of each RSF. A sample was taken for histological and immunohistochemistry studies, fibroblasts, MCs, angiogenesis, collagen bundle density, and the presence of vascular endothelial growth factor (VEGF)+ cells. An additional sample was taken to evaluate the flap's incision strength. Treatment with BMMSCs (17.8 ± 0.37) significantly increased RSF survival compared with the control (13.3 ± 0.35) and placebo (16.1 ± 0.27) groups (one-way analysis of variance, P = .000; least significant difference, P = .000, P = .002). There was a significant improvement in angiogenesis, as confirmed by stereologic examination. Assessment of VEGF+ cells showed prominent neovascularization in BMMSC-treated RSFs compared with the control and placebo groups. Subdermal injection of BMMSC significantly increased ischemic RSF survival as a result of stimulated neovascularization in T1DM rats. Treatment of diabetic RSF with BMMSCs showed no beneficial effects in the fibroblast number and biomechanical parameters for the repair of ischemic wounds in the rat model. Treatment with BMMSCs significantly increased collagen bundle density.