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.     

延边黄牛背最长肌差异表达基因的筛选、克隆及序列分析

应用引物复性控制技术筛选肌内脂肪含量差异极显著的延边黄牛背最长肌组织差异表达基因,寻找与肌内脂肪沉积的相关候选基因。文章选取30头28月龄延边黄牛阉牛的背最长肌组织测定肌内脂肪含量,选取肌内脂肪含量差异极显著的最高和最低各3头组成RNA池,采用引物复性控制技术,分析了两组个体背最长肌组织差异表达基因。利用20对随机引物差异显示扩增下,共获得12条ESTs(片段大小为200～890 bp),其中8个为已知的ESTs分别与细胞骨架形成、细胞因子信号转导、蛋白质合成、能量代谢和其他功能的差异基因,4个未知的ESTs。结果表明,应用引物复性控制技术筛选得到了12个可能参与了肌内脂肪沉积调控的ESTs,为进一步筛选肌内脂肪沉积相关的基因奠定了基础。     

rhBMP-2诱导异位软骨修复重建兔气管缺损的实验研究

目的：探讨重组人骨形成蛋白-2（rhBMP-2）作为激活物诱导异位软骨修复并重建免气管缺损的可行性。方法：取24只新西兰大白兔,制备气管前壁软骨1／3缺损模型。随机分为A、B组,每组12只,A组为实验组,在气管缺损处前壁颈前肌肉修补,多点注射rhBMP-2;B组于气管软骨缺损部位直接颈前肌群修补。术后观察动物一般情况,于4、8、12周取材进行大体观察、HE染色观察重建区域情况。结果：术后A组动物均存活至实验完成,B组因气道感染及气道分泌物堵管潴留致使实验兔死亡,其余动物出现皮下气肿,呼吸不畅等情况。组织学观察A组有明显的新生软骨细胞及少量软骨样组织,可见结缔组织包绕,周围肌肉组织完整,排列整齐,未见明显坏死组织,有少量淋巴细胞浸润。B组未见软骨组织生成,可见大量肉芽组织增生,结缔组织排列紊乱,伴少量坏死组织,大量淋巴浸润。结论：rhBMP-2可通过注射到颈前肌肉修补肌群中诱导软骨细胞和软骨样组织生成,减轻炎症反应,联合颈前肌瓣修复重建气管缺损能充分维持修复重建后的气道形态,具有减少术后皮下气肿、气管狭窄的作用,有望用于临床修复重建气管纽织缺损。     

Reflex control of posterior shoulder muscles from arm afferents in healthy people

In order to position the hand during functional tasks, control of the shoulder is required. Heteronymous reflexes from the upper limb to shoulder muscles are used to assist in this control. To investigate this further, the radial and ulnar nerves were stimulated at elbow level whilst surface electromyographic activity of posterior deltoid, infraspinatus and latissimus dorsi muscles were recorded. In addition, the cutaneous branch of the radial nerve and the skin of the fifth digit were stimulated in order to investigate any cutaneous contribution to reflex activity. Reflexes were evoked in all three of these shoulder muscles from hand and/or forearm afferents. However, the reflexes differed; whereas both excitatory and inhibitory reflexes were evoked in posterior deltoid and infraspinatus, the reflexes in latissimus dorsi were mainly excitatory. Cutaneomuscular reflexes were seldom evoked here, but when they were present they were generally evoked at longer latencies than the reflexes evoked by mixed nerve stimulation. The results suggest a role for reflexes originating from the forearm and/or hand in the control of the shoulder.     

Dynamics of enzymatic interactions during short flap human Okazaki fragment processing by two forms of human DNA polymerase δ

Lagging strand DNA replication requires the concerted actions of DNA polymerase δ, Fen1 and DNA ligase I for the removal of the RNA/DNA primers before ligation of Okazaki fragments. To better understand this process in human cells, we have reconstituted Okazaki fragment processing by the short flap pathway in vitro with purified human proteins and oligonucleotide substrates. We systematically characterized the key events in Okazaki fragment processing: the strand displacement, Pol δ/Fen1 combined reactions for removal of the RNA/DNA primer, and the complete reaction with DNA ligase I. Two forms of human DNA polymerase δ were studied: Pol δ4 and Pol δ3, which represent the heterotetramer and the heterotrimer lacking the p12 subunit, respectively. Pol δ3 exhibits very limited strand displacement activity in contrast to Pol δ4, and stalls on encounter with a 5′-blocking oligonucleotide. Pol δ4 and Pol δ3 exhibit different characteristics in the Pol δ/Fen1 reactions. While Pol δ3 produces predominantly 1 and 2 nt cleavage products irrespective of Fen1 concentrations, Pol δ4 produces cleavage fragments of 1–10 nts at low Fen1 concentrations. Pol δ3 and Pol δ4 exhibit comparable formation of ligated products in the complete system. While both are capable of Okazaki fragment processing in vitro, Pol δ3 exhibits ideal characteristics for a role in Okazaki fragment processing. Pol δ3 readily idles and in combination with Fen1 produces primarily 1 nt cleavage products, so that nick translation predominates in the removal of the blocking strand, avoiding the production of longer flaps that require additional processing. These studies represent the first analysis of the two forms of human Pol δ in Okazaki fragment processing. The findings provide evidence for the novel concept that Pol δ3 has a role in lagging strand synthesis, and that both forms of Pol δ may participate in DNA replication in higher eukaryotic cells.