Just an additional hydrogen bond can dramatically reduce the catalytic activity of Bacillus subtilis lipase A I12T mutant: An integration of computational modeling and experimental analysis

Understanding the structural basis and energetic property of hydrogen bonding and its effects on enzymatic activity is fundamentally important for the rational design of specific enzymes with desired biological functions. In the current study, site-directed mutagenesis analysis preliminarily revealed that the amino acid substitution of Ile12 with Thr12 (I12T) dramatically reduced the hydrolytic activity of Bacillus subtilis lipase A. A further computational investigation proposed that the I12T mutation would establish a geometrically perfect hydrogen bond between the mutated Thr12 and catalytic Ser77 of lipase A, which considerably impaired the catalytic capability of lipase A through two distinct but complementary approaches: rigidizing the enzyme active site and lowering the nucleophilic ability of the catalytic residue Ser77. To verify this hypothesis, a homogenous mutation I12S serving as the control to the I12T mutation was created to examine the hydrogen bonding effect on enzymatic activity. It was found that the I12S mutant only suffered from a slight damage in its hydrolytic ability due to absence of the hydrogen bond originally present at the Thr12–Ser77 interface in the I12T mutant, which was further characterized systematically by quantum mechanics/molecular mechanics (QM/MM) modeling, atom-in-molecules (AIM) analysis and molecular dynamics (MD) simulation. It is suggested that the hydrogen bond arising from the I12T mutation in lipase A can considerably reduce the flexibility and mobility of the enzyme active site, thus impairing the catalytic activity of the lipase A I12T mutant remarkably; the activity loss can be, however, largely recovered by replacing Thr residue at the 12th position of I12T mutant with its analog Ser, which is chemically similar to Thr but cannot form effective hydrogen bonding with Ser77.     

Lithium di silicate ceramic surface treated with Er,Cr:YSGG and other conditioning regimes bonded to orthodontic bracket

AimTo assess bond integrity and modes of failure of metallic brackets to lithium disilicate ceramics (LDC) conditioned with Er,Cr:YSGG laser (ECL).Material and methodsSixty LDC were arbitrarily allocated into six groups (n = 15) according to the type of ceramic surface conditioning treatment. Group 1 surface treated with silane (S) only, group 2 surface etched with hydrofluoric acid (HF)+ S, group 3 surface conditioned with HF+ ultrasonic bath (UB)+ S, group 4 sand blasting (SB) of glass ceramic surface with 50 µm Al₂O₃, group 5 surface conditioned with self-etch ceramic primer (SECP) and in group 6 surface treated with ECL + S. After conditioning, the specimens were positioned in a universal testing device for shear bond strength (SBS) testing. Adhesive Remnant Index (ARI) was used to determine sites of bond failure. Among experimental groups analysis of variance (ANOVA) and Tukey multiple comparison test was used at a significance level of (p < 0.05).ResultsThe highest SBS values were observed in group 3 HF+ UB + S (18.21 ± 1.241) and the lowest SBS values were displayed group 1 surface treated with S only (5.21 ± 0.23). Specimens surface conditioned in group 2 with HF+ S (17.85 ± 1.25), group 3 HF+ UB + S (18.21 ± 1.241) and group 6 ECL + S (17.09 ± 1.114) unveiled comparable SBS values (p > 0.05).ConclusionLDC conditioned with ECL at (4.5 W and 30 Hz) has a potential to be used in clinical settings alternate to HF acid.     

酪蛋白磷酸多肽钙磷复合体的应用顺序对乳牙牙本质粘结强度的影响

目的探讨酪蛋白磷酸多肽钙磷复合体(casein phosphopetide-amorphic calcium phosphat, CPP-ACP)应用顺序对乳牙牙本质粘结强度及对乳牙牙本质小管封闭作用的影响。方法选取人56颗无龋乳磨牙,磨除牙合面釉质,制备牙本质平面。将其中48颗乳磨牙随机分3大组(n=16)。A组:仅酸蚀15 s。B组:涂CPP-ACP后酸蚀15 s。C组:酸蚀15 s后涂CPP-ACP。各组分为2小组(n=8),其中AN,BN,CN组使用Prime&Bond NT(NT)全酸蚀粘结剂,AS,BS,CS组使用Single bond 2(SB2)全酸蚀粘结剂。制作树脂粘结试件,电子万能试验机测定剪切强度(SBS值)。其余8个乳磨牙随机分成4组(n=2),分别为空白组(S1组)、酸蚀组(S2组)、酸蚀后涂CPP-ACP组(S3组)、涂CPP-ACP后酸蚀组(S4组)分别用扫描电镜观察粘结前牙本质小管封闭情况。结果剪切实验结果为AN组、BN组、CN组剪切强度无统计学差异(P>0.05);AS组,BS组,CS组剪切强度也无统计学差异(P>0.05);AN组>AS组,BN组>BS组,CN组>CS组差异有统计学意义(P<0.05)。扫描电镜结果为S3组中牙本质小管封闭数目最多。结论在酸蚀后应用CPP-ACP处理乳牙牙本质不影响NT和SB2两种全酸蚀粘结剂的粘结强度;可以达到有效的牙本质小管封闭;Prime&Bond NT(NT)全酸蚀粘结系统的粘结强度高于Single bond 2(SB2)。     

EDTA对年轻恒牙釉质粘接强度的影响

目的 研究乙二胺四乙酸二钠（EDTA）对年轻恒牙釉质粘接强度的影响。方法 收集手术拔除的牙根未发育完全的上下颌第三磨牙,共180颗,选取全部离体牙牙冠颊面与牙长轴相对平行的釉质面（倾斜角度小于5°）作为实验粘接面,按实验方法不同随机均分为5组,每组36颗,分别进行以下处理：G0（对照组）：37%磷酸常规酸蚀;G1：37%磷酸+17% EDTA;G2：37%磷酸+10% EDTA;G3：17% EDTA+37%磷酸,G4：10% EDTA+37%磷酸。以上样本均采用GLUMA通用型粘接剂粘接后,行流动树脂堆砌。使用万能材料试验机进行树脂剪切粘接强度的测试。剔除剪切测试中由于树脂块断裂而未形成树脂-釉质面分离的试件,针对剪切粘接强度采用SPSS21.0统计软件进行统计学分析。结果 G0的平均剪切粘接强度（9.80±2.78 MPa）要明显低于G1（11.56±3.30 MPa）、G2（12.01±3.28 MPa）和G4（12.19±3.04 MPa）的剪切粘接强度,其差异都具有统计学意义（P<0.05）。尽管G3的平均剪切粘接强度（10.75±2.85 MPa）要高于G0,但两组之间的强度差异无统计学意义（P=0.261）。此外,G1、G2、G3和G4各组之间的平均剪切粘接强度差异无统计学意义（P>0.05）。结论：酸蚀前、后EDTA去蛋白化处理较单纯酸蚀处理更能增强树脂的剪切粘接强度,但其作用可能会受到EDTA浓度的影响。     

不同表面处理方法在纳米混合树脂复合材料修复中的应用

目的： 分析不同表面处理方法和粘接剂自酸蚀功能单体对树脂-复合材料界面即时修复粘结强度和完整性的影响。方法： 采用纳米树脂复合材料制作98个树脂复合材料,随机分为A1、A2、B1、B2、C、D组,各14个试件。表面未处理的试件作为阳性对照组（14个试件）。A1组用Gluma 通用粘接剂系统抛光,A2组用Gluma 通用粘接剂系统抛光、喷砂,B1组用Tokuyama Bond ForceⅡ^TM粘结系统抛光,B2组用Tokuyama Bond ForceⅡ^TM抛光、喷砂,C组仅经抛光样品组。D组仅做喷砂。采用与底物相同的树脂复合材料,对修复后试件进行剪切粘结强度（shear bond strength,SBS）测试,所有样本均进行电子显微镜扫描、测定表面轮廓,进行失效分析。采用SPSS 20.0软件包对数据进行统计学处理。结果： D组修复粘结强度显著高于阴性对照组（P<0.05）,A1、A2、B2、B1组粘结强度显著高于C、D组（P<0.05）;B1、D或A1组相比,粘结强度无显著差异（P>0.05）;B2组、阳性对照组粘结强度无显著差异（P>0.05）。除喷砂、TBFⅡ外,阳性对照组粘合强度值显著高于A1、C组（P<0.05）。抛光后表面粘合失效率高于喷砂样本（P<0.05）;抛光、Gluma处理样品粘合失败率高于抛光、TBFⅡ处理样品（P<0.05）;喷砂、TBFⅡ处理的表面内聚破坏率高于抛光、TBFⅡ处理（P<0.05）。抛光技术的表面粗糙度与喷砂技术相比,较规则且粗糙度较低（P<0.05）。结论： 经喷砂处理的复合材料基材加TBFⅡ,其修复粘结性最强,且表面内聚破坏率较高,TBFⅡ处理粘合失败率低。但经喷砂处理后的材料易堆积食物残渣,而抛光后的材料则不易发生。使用喷砂处理的复合材料基材上加TBFⅡ的患者,需正确有效地维护口腔卫生。     

Adhesion procedures for CAD/CAM indirect resin composite block: A new resin primer versus a conventional silanizing agent

PurposeThe purpose of this study was to evaluate the effectiveness of both a resin primer containing methyl methacrylate (MMA) and a silanizing agent on bonding to indirect resin composite blocks, using two types of build-up hybrid resin composites.MethodsSHOFU BLOCK HC (Shofu) specimens were blasted with alumina, after which one of two surface treatments was applied: CERA RESIN BOND (Shofu, the Silane group) or HC primer (Shofu, the MMA group). Resin composites made using either Solidex Hardura (SDH, Shofu) or Ceramage Duo (CMD, Shofu) were built up and micro-tensile bond strength (μTBS) values were measured after storage in water for either 24 h or 6 months (n = 24 per group). The fracture surfaces after μTBS measurements and the resin block/build-up resin interfaces were observed by scanning electron microscopy (SEM).ResultsThe bond strength of the Silane/SDH group significantly decreased after 6 months (p < 0.001), whereas in the MMA group there was no significant loss after 24 h or 6 months (p = 0.99). In the CMD group, the bond strength after 6 months was significantly lowered in both the Silane group (p < 0.001) and the MMA group (p < 0.001), but the latter still showed greater adhesion. SEM images demonstrated that the matrix resin was partially destroyed at the fracture surfaces of the MMA group and fracture surface unevenness was observed.ConclusionsA primer containing MMA produced stronger bonding to CAD/CAM resin even after long-term aging compared to a silane treatment.     

Alpha,beta hybrid peptides: a polypeptide helix with a central segment containing two consecutive beta-amino acid residues

Conformational studies on the synthetic 11-aa peptide t-butoxycarbonyl (Boc)-Val-Ala-Phe-alpha-aminoisobutyric acid (Aib)-(R)-beta3-homovaline (betaVal)-(S)-beta3-homophenylalanine (betaPhe)-Aib-Val-Ala-Phe-Aib-methyl ester (OMe) (peptide 1; betaVal and betaPhe are beta amino acids generated by homologation of the corresponding l-residues) establish that insertion of two consecutive beta residues into a polypeptide helix can be accomplished without significant structural distortion. Crystal-structure analysis reveals a continuous helical conformation encompassing the segment of residues 2-10 of peptide 1. At the site of insertion of the betabeta segment, helical hydrogen-bonded rings are expanded. A C15 hydrogen bond for the alphabetabeta segment and two C14 hydrogen bonds for the alphaalphabeta or betaalphaalpha segments have been characterized. The following conformational angles were determined from the crystal structure for the beta residues: betaVal-5 (= -126 degrees, = 76 degrees, and psi = -124) and betaPhe-6 (=-88 degrees, = 80 degrees, and psi =-118). The N terminus of the peptide is partially unfolded in crystals. The 500-MHz 1H-NMR studies establish a continuous helix over the entire length of the peptide in CDCl3 solution, as evidenced by diagnostic nuclear Overhauser effects. The presence of seven intramolecular hydrogen bonds is also established by using solvent dependence of NH chemical shifts.     

Shear bond strength of orthodontic brackets to enamel after application of a caries infiltrant

Objective:To examine differences in the shear bond strength of orthodontic brackets on differently mineralized enamel surfaces after applying a caries infiltrant or conventional adhesive.Materials and Methods:A total of 320 bovine incisors were assigned to eight pretreated groups, and the shear force required for debonding was recorded. Residual adhesive was evaluated by light microscopy using the adhesive remnant index. Statistical analysis included Kolmogorov-Smirnov, analysis of variance (ANOVA), and Scheffé tests.Results:The highest bond strength (18.8 ± 4.4 MPa) was obtained after use of the caries infiltrant. More residual adhesive and fewer enamel defects were observed on infiltrated enamel surfaces. Brackets on demineralized enamel produced multiple enamel defects.Conclusions:Acceptable bond strengths were obtained with all material combinations. A caries-infiltrant applied before bracket fixation has a protective effect, especially on demineralized enamel.