Interchain hydrophobic clustering promotes rigidity in HIV-1 protease flap dynamics: new insights from Molecular Dynamics

The dynamics of HIV-1 protease (HIV-pr), a drug target for HIV infection, has been studied extensively by both computational and experimental methods. The flap dynamics of HIV-pr is considered to be more important for better ligand binding and enzymatic actions. Moreover, it has been demonstrated that the drug-induced mutations can change the flap dynamics of HIV-pr affecting the binding affinity of the ligands. Therefore, detailed understanding of flap dynamics is essential for designing better inhibitors. Previous computational investigations observed significant variation in the flap opening in nanosecond time scale indicating that the dynamics is highly sensitive to the simulation protocols. To understand the sensitivity of the flap dynamics on the force field and simulation protocol, molecular dynamics simulations of HIV-pr have been performed with two different AMBER force fields, ff99 and ff02. Two different trajectories (20?ns each) were obtained using the ff99 and ff02 force field. The results showed polarizable force field (ff02) make the flap tighter than the nonpolarizable force field (ff99). Some polar interactions and hydrogen bonds involving flap residues were found to be stronger with ff02 force field. The formation of interchain hydrophobic cluster (between flap tip of one chain and active site wall of another chain) was found to be dominant in the semi-open structures obtained from the simulations irrespective of the force field. It is proposed that an inhibitor, which will promote this interchain hydrophobic clustering, may make the flaps more rigid, and presumably the effect of mutation would be small on ligand binding.     

火球菌Pyrococcus furious瓣状核酸内切酶1的表达纯化及酶学特征

【目的】克隆表达和纯化火球菌Pyrococcus furious来源的瓣状核酸内切酶1基因pFEN1(PF1414),对该蛋白的活性和酶学特征进行鉴定和分析。【方法】将pFEN1在大肠杆菌中进行重组表达,经亲和层析纯化得到电泳纯蛋白;利用人工合成的荧光标记的寡核苷酸片段作为底物,用变性聚丙烯酰胺凝胶电泳鉴定pFEN1在体外的酶学特性以及与其他蛋白的相互作用。【结果】pFEN1重组蛋白能在大肠杆菌中进行高效表达;高于100 mmol/L的NaCl会抑制pFEN1的活性;pFEN1的核酸酶活性依赖于金属离子Mg~(2+)或Mn~(2+),且Mn~(2+)的催化效率优于Mg~(2+);来自嗜热古菌的pFEN1是一种耐高温蛋白,最适反应温度为60–65°C;增殖细胞核抗原(PCNA)能促进pFEN1的内切酶活性。【结论】本研究证实pFEN1是一种Mg~(2+)或Mn~(2+)依赖的核酸内切酶,且PCNA能促进该酶的活性。     

负压封闭引流技术在腹部带蒂皮瓣供瓣区的应用

目的:探讨负压封闭引流(vaccumsealingdrainage,VSD)技术在腹部带蒂皮瓣供瓣区中的治疗作用。方法:10例上肢深度创面大小为9×6 cm-14×9 cm,创面清创后行腹部带蒂皮瓣移植,皮瓣面积10×7 cm-20×15 cm,供瓣区应用VSD覆盖,医用半透性贴膜封闭整个术区,连接中心负压,持续低负压吸引,每日外用1000 mL生理盐水持续冲洗VSD。约3周后行皮瓣断蒂术,供瓣区植皮。结果:10例皮瓣全部存活,1例患者术后当天引流区域出现疼痛,经止痛治疗后未再出现。1例患者术后2周出现VSD堵塞,经处理后恢复正常引流。无VSD装置更换病例。供瓣区周边皮肤无浸渍及感染现象,VSD撤除后见基底肉芽组织生长新鲜,创面均无感染,移植皮片存活。结论:VSD技术可以有效保护供瓣区,刺激肉芽生长,为后期皮片移植闭合创面提供了良好条件。VSD技术可安全有效的应用于腹部带蒂皮瓣供瓣区。     

Tissue engineering of bone: the reconstructive surgeon's point of view

Bone defects represent a medical and socioeconomic challenge. Different types of biomaterials are applied for reconstructive indications and receive rising interest. However, autologous bone grafts are still considered as the gold standard for reconstruction of extended bone defects. The generation of bioartificial bone tissues may help to overcome the problems related to donor site morbidity and size limitations. Tissue engineering is, according to its historic definition, an "interdisciplinary field that applies the principles of engineering and the life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function". It is based on the understanding of tissue formation and regeneration and aims to rather grow new functional tissues than to build new spare parts. While reconstruction of small to moderate sized bone defects using engineered bone tissues is technically feasible, and some of the currently developed concepts may represent alternatives to autologous bone grafts for certain clinical conditions, the reconstruction of large-volume defects remains challenging. Therefore vascularization concepts gain on interest and the combination of tissue engineering approaches with flap prefabrication techniques may eventually allow application of bone-tissue substitutes grown in vivo with the advantage of minimal donor site morbidity as compared to conventional vascularized bone grafts. The scope of this review is the introduction of basic principles and different components of engineered bioartificial bone tissues with a strong focus on clinical applications in reconstructive surgery. Concepts for the induction of axial vascularization in engineered bone tissues as well as potential clinical applications are discussed in detail.     

Comprehensive mapping of the C-terminus of flap endonuclease-1 reveals distinct interaction sites for five proteins that represent different DNA replication and repair pathways

Flap endonuclease-1 (FEN-1) is a multifunctional and structure-specific nuclease that plays a critical role in maintaining human genome stability through RNA primer removal, long-patch base excision repair, resolution of DNA secondary structures and stalled DNA replication forks, and apoptotic DNA fragmentation. How FEN-1 is involved in multiple pathways, of which some are seemingly contradictory, is of considerable interest. To date, at least 20 proteins are known to interact with FEN-1; some form distinct complexes that affect one or more FEN-1 activities presumably to direct FEN-1 to a particular DNA metabolic pathway. FEN-1 consists of a nuclease core domain and a C-terminal extension. While the core domain harbors the nuclease activity, the C-terminal extension may be important for protein-protein interactions. Here, we have truncated or mutated the C-terminus of FEN-1 to identify amino acid residues that are critical for interaction with five proteins representing roles in different DNA replication and repair pathways. We found with all five proteins that the C-terminus is important for binding and that each protein uses a subset of amino acid residues. Replacement of one or more residues with an alanine in many cases leads to the complete loss of interaction, which may consequently lead to severe biological defects in mammals.     

颏下岛状皮瓣修复口腔颌面部肿瘤术后缺损60 例分析

目的:对颏下岛状皮瓣修复口腔颌面部肿瘤术后缺损患者进行观察与探讨,为临床应用颏下岛状皮瓣修复治疗存在口腔颌面部肿瘤术后缺损情况的患者提供依据。方法:对2013年6月到2015年6月期间在医院接受颏下岛状皮瓣修复治疗的60例口腔颌面部肿瘤术后缺损患者进行临床回顾性研究,按照是否存在颈淋巴结转移分为两组进行研究比较。结果:研究组口腔颌面部肿瘤术后缺损患者的颏下岛状皮瓣存活率,术后复发率,术后6个月、1年、2年的生存率,术后并发症总发生率,总满意度与对照组口腔颌面部肿瘤术后缺损患者相比,组间差异不明显(P0.05),不具有统计学意义。结论:使用颏下岛状皮瓣修复治疗口腔颌面部肿瘤术后缺损患者的效果较好,大多数患者对于临床修复治疗效果比较满意。而且,对存在颈淋巴结转移的患者实施颈部淋巴结的清扫术及术后放疗对于临床疗效的影响不大,基本不影响皮瓣的存活,具有重要的临床应用价值。     

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.     

三种不同皮瓣修复口腔颌面组织缺损的临床效果

目的:探讨三种皮瓣在口腔面部组织缺损修复中的临床效果。方法:以2014年1月到2014年12月在我院皮瓣修复口腔面部组织缺损患者64例为研究对象。胸大肌皮瓣26例,前臂皮瓣22例,股前外侧皮瓣16例。术后3~12月对患者临床疗效进行观察评价。结果:患者均采用胸大肌皮瓣、股前外侧皮瓣和前臂皮瓣进行口腔面部组织缺损的修复,不同皮瓣来源数量组间比较差异均无统计学意义(P0.05)。患者术后均治愈,移植成功率100%,组间比较差异无统计学意义(P0.05)。少数患者发生积血、感染不良反应情况,总不良反应发生率为12.5%,不同组间的不良反应发生率比较差异无统计学意义(P0.05)。结论:三种不同的皮瓣修复口腔面部组织缺损临床效果均较好,皮瓣的选择较广泛,显著改善了患者口腔生理功能,提高了患者的生活质量,临床推广使用价值高。     

新型小鼠跨区供血耳瓣模型的构建

目的：建立一个实时活体观察血管形态学变化小鼠跨区供血耳瓣模型。方法体重25～30 g清洁级ICR小鼠30只,双耳脱毛后,观察其血管分布情况。小鼠麻醉后,用眼科剪从尾侧向头侧剪断鼠耳基底部尾侧2/3,保留头侧1/3,形成耳前血管蒂跨三个血管体、二个choke区的耳瓣模型。将小鼠侧卧置于二维图像采集系统的动物承载台上,调节体视显微镜物镜并固定为25倍,设置步进参数,“弓”型路线渐次、局部采集造模后0,1,2,3,5,7,10,14,21,30 d的时间点图像,合成鼠耳全景图。重点观察皮瓣的坏死率、皮瓣内choke血管的形态学变化。结果 ICR小鼠耳有三个恒定的血管体来供养,从内到外依次为头侧血管体、中间血管体及尾侧血管体。术后5 d,耳瓣坏死面积趋于稳定,坏死率为（15±7）％。内侧血管体与中间血管体之间的choke动静脉的管径出现快速扩增,两者都在第10天左右达最大,choke静脉管径最高峰可达到原来的（3.9±0.5）倍,choke动脉管径最高峰可达到原来的（3.5±0.7）倍。10 d后,choke静脉管径开始减小,21 d后逐渐平稳,而choke动脉管径于术后10 d左右开始平稳,之后无明显减小。结论①跨区皮瓣切取后,静脉扩张是被动扩张,而动脉扩张是主动增值;②跨区皮瓣切取后血流动力学供区与潜力供区之间的choke区参与扩张的choke血管数量及扩张度均小于解剖供区与血流动力学供区之间的choke血管;③小鼠耳瓣模型为研究血管扩张机制及遴选促皮瓣存活药物的理想动物模型。