Spermine as a possible endogenous allosteric activator of carboxypeptidase A: multispectroscopic and molecular simulation studies

Abstract

Carboxypeptidase A (EC.3.4.17.1) is a zinc-containing proteolytic enzyme that removes the C-terminal amino acid from a peptide chain with the free carboxylate-terminal. In this study, the effect of spermine interaction on the structure and thermal stability of Carboxypeptidase A was investigated by ultraviolet???visible spectroscopy, fluorescence spectroscopy, circular dichroism, Kinetic measurement, molecular docking and simulation studies have also been followed at the pH of 7.5. The transition temperature of Carboxypeptidase A, as a criterion of protein thermal stability, in the presence of spermine was enhanced by increasing the concentration of spermine. The results of fluorescence intensity changes, at two temperatures of 308 and 318?K, also suggested that spermine had a great ability to quench the fluorescence of Carboxypeptidase A through the static quenching procedure. The thermodynamic parameters changes, including standard Gibbs free-energy, entropy and enthalpy, showed that the binding of spermine to Carboxypeptidase A was spontaneous and the hydrogen bonding and van der Waals interactions played a major role in stabilizing the Carboxypeptidase A–spermine complex. The changes in the content of the α-helix and the β-sheet of the Carboxypeptidase A with binding to spermine were shown by the CD spectra method. Further, kinetic studies revealed that by increasing concentration of spermine, the activity of Carboxypeptidase A was enhanced. Also, the docking study revealed that the hydrogen bonding and van der Waals interactions played a major role in stabilizing the Carboxypeptidase A–spermine complex. As a result, spermine could be considered as an activator and a stabilizer for Carboxypeptidase A.

Communicated by Ramaswamy H. Sarma     

Optogenetic reporters

The discovery of naturally evolved fluorescent proteins and their subsequent tuning by protein engineering provided the basis for a large family of genetically encoded biosensors that report a variety of physicochemical processes occurring in living tissue. These optogenetic reporters are powerful tools for live‐cell microscopy and quantitative analysis at the subcellular level. In this review, we present an overview of the transduction mechanisms that have been exploited for engineering these genetically encoded reporters. Finally, we discuss current and future efforts towards the combined use of various optogenetic actuators and reporters for simultaneously controlling and imaging the physiology of cells and tissues.     

Multinational study of oestrogen and progesterone receptor immunocytochemistry on breast carcinoma fine needle aspirates

?. P. Marin?ek, N. Nolde, I. Kardum‐Skelin, R. Nizzoli, B. Önal, T. Rezanko, E. Tani, K. T. Ostovi?, P. Vielh, F. Schmitt and G. Kocjan
Multinational study of oestrogen and progesterone receptor immunocytochemistry on breast carcinoma fine needle aspirates Objectives: To collect data on the variability of immunocytochemical (ICC) procedures used to detect oestrogen/progesterone receptors (ER/PR) on cytological material; to test the reproducibility of results; and to identify the crucial points in the ICC procedures that affect the result. Methods: Ten laboratories from eight countries participated in a two‐part study. In the first part, one of the participants (the coordinator) prepared and distributed cytospins from a fine needle aspirate of a primary breast carcinoma. Laboratories performed ICC staining for ER/PR according to their own methods on the test slides and in‐house positive controls. Slides were returned to the coordinator together with information on the preparation of positive control slides and the ICC methodology used. In the second part, obligatory methods of fixation and antigen retrieval were specified. Evaluation of results included grading the number of positive cells, staining intensity, background staining, cytoplasmic staining, sample condition and cellularity. Participants evaluated their own results, which were subsequently evaluated by the coordinator. Results: There was great variability in the preparation of slides for in‐house controls and ICC methodology. The outcome of ICC staining of in‐house control slides was excellent in two laboratories, adequate in three, sub‐optimal in four and inadequate in one. Only six obtained a positive reaction on the test slides and not all were of a high quality. Results of the second run were greatly improved in terms of cellularity of in‐house positive control slides, and scores for the percentage of stained cells and staining intensity of control and test slides. Cytospins and monolayer (ThinPrep^®) preparations were superior to direct smears; methods of fixation and antigen retrieval were the key points in the staining process. Conclusions: Our experience points to the need for guidelines for hormonal receptor determination and external quality control on cytological material, in order for cytological methods to be used in routine clinical practice with a suitable degree of confidence.     

小切口微创手术治疗新鲜跟腱断裂的临床价值

目的：探讨小切口微创手术治疗新鲜跟腱断裂的一晦床价值。方法：选取我院新鲜闭合性跟腱断裂患者50例,随机分为实验组和对照组各25例。实验组行小切口手术,对照组行常规切口手术。术后对患者进行随访,采用美国足踝协会（AOFAS）推荐的评分标准对患者术后功能恢复情况进行评价,观察并记录完全恢复患者例数、完全恢复时间、小腿最大周长差和术后并发症发生情况。结果：实验组AOFAS评分为（98．6±9．7）分,痊愈率96．00％,痊愈时间（20．2±3．2）周,两侧小腿最大周长差为（0．79＋0．68）cm,共有1例患者出现并发症,并发症发生率8％;而对照组的AOFAS评分为（91．4±11．5）分,痊愈率92．00％,痊愈时间（22．4±3．8）周,两侧小腿最大周长差为（0．91~0．76）cm;共有6例患者出现并发症,并发症的发生率为24％。两组患者的痊愈率、两侧小腿最大周长差比较差异无统计学意义（痊愈率：x2=-0．355,P=0．552;侧小腿最大周长差：t=O．588,P=0．559）;而与对照组比较,实验组AOFAS评分明显升高,完全恢复时间明显缩短,术后并发症的发生率显著降低,差异均有统计学意义（AOFAS评：t=2．393,P=0．021;恢复时间：t=2．150,P=0．037;并发症发生率：xⅫ．153,P=0．042）。结论：小切口手术与常规切口手术治疗新鲜跟腱断裂的疗效相当,但小切口手术术后恢童时间曼短．并发症更少．临床价值相对更高．     

Outcome of stand-alone thoracoscopic epicardial left atrial posterior box isolation with bipolar radiofrequency energy for longstanding persistent atrial fibrillation

Introduction

Catheter ablation of longstanding (> 1 year) persistent atrial fibrillation (AF) is associated with poor outcome. This might be due to remodelling and fibrosis formation, mainly located in the posterior left atrial (LA) wall. Therefore, we adopted a thoracoscopic epicardial box isolation of the posterior left atrium using bipolar RF energy with intraoperative testing of conduction block.

Methods and results

Bilateral thoracoscopic box isolation was performed with a bipolar RF clamp. Entrance block was defined as absence of a conducted electrogram within the box, while exit block was confirmed by pacing at 10.0 V/2 ms. Ablation outcome was evaluated after 3, 6, 12 and 24 months with 12-lead ECGs and 24-hour Holter recordings.Twenty-five consecutive patients were included (58 ± 7 years, persistent AF duration 1.8 ± 0.9 years). Entrance block was achieved in all patients and exit block confirmed if sinus rhythm was achieved. After 17 ± 7 months, 76 % of the patients (n = 19) were free of AF recurrence. One patient died within 1 month and was considered an ablation failure. Four patients with AF recurrences regained sinus rhythm with additional catheter ablation or antiarrhythmic drugs.

Conclusions

Treatment of longstanding persistent AF with thoracoscopic epicardial LA posterior box isolation using bipolar RF energy with intraoperative testing of conduction block is feasible and highly effective.     

网络云交互技术辅助外科实践教学的创新应用探索

目的:探索传统教学模式结合网络云交互技术在外科实践教学中的应用效果。方法:于我校2011级五年制临床医学专业本科生中选取76人,随机平均分为两组,分别采取单纯传统大课教学模式、传统教学结合网络云交互技术教学模式;课程结束后进行闭卷理论考试和实践操作考试,综合两项成绩进行评定。同时向两组学员发放调查问卷,以此评价使用网络云交互技术辅助教学的效果。结果:采取传统教学结合网络云交互技术教学模式组的成绩优于采取单纯传统教学模式组,两组成绩差异具有统计学意义(P0.05)。结论:在传统教学模式的基础上添加网络云交互技术更能激发学生的学习兴趣,大大提高了学习效率,更有利于本科生外科实践的教学以及教学资源的整合及教学档案的建立。     

空气微生物群落解析方法:从培养到非培养

随着世界范围内流行性疾病以及我国空气雾霾事件的不断发生,空气生物性污染的研究开始受到高度重视,其研究方法也随着分子生物学技术的快速发展而不断更新,由早期以生化技术为基础的研究方法转变为以现代分子生物学技术为基础的研究方法。综述了空气微生物群落多样性解析方法从培养到非培养的发展过程,包括培养技术法、BIOLOG技术、生物标记法、基因指纹图谱技术、核酸杂交技术、实时荧光定量PCR、空气微生物宏基因组学及基因芯片技术,阐述了这些技术的基本原理,比较了各种技术的优缺点并重点介绍了它们在空气微生物群落多样性研究中的应用概况,最后展望了空气微生物学研究的发展方向。     

DNA binding studies of antibiotic drug cephalexin using spectroscopic and molecular docking techniques

The purpose of this study was to explore an accurate characterization of the binding interaction of antibiotic drug cephalexin with calf thymus DNA (CT-DNA) as a relevant biological target by using UV absorption, fluorescence spectroscopy and circular dichroism (CD) in vitro under simulated physiological conditions (pH = 7.4) and also through a molecular modeling study. The results showed that the drug interacts with the DNA helix via a minor groove binding mode. The thermodynamic parameters were calculated and showed that the reaction between the drug and CT-DNA was exothermic. In addition, the drug enforced traceable changes in the viscosity of DNA. The molecular modeling results indicated that cephalexin forcefully binds to the minor groove of DNA with a relative binding energy of ?21.02?kJ mol^?1. The obtained theoretical results were in good agreement with those obtained from experimental studies.     

Comparative study on different therapies on patients with primary central nervous system lymphoma

Objective

To compare and analyze three therapies on patients with primary central nervous system lymphoma (PCNSL), aiming to provide evidences for future treatment and prognosis.

精细化管理在病理学技术质量控制中的应用

精准医疗是近年来医学的发展方向,精准医疗依赖于精准的诊断,而精准诊断则需要高质量病理学切片技术作为支撑。武汉大学人民医院病理科将精细化管理理念融入病理学技术质控和管理工作的每一环节,通过采用不同颜色包埋框对组织分类、时间梯度法调控出片时间、根据人员资质及医疗风险对病理学技术人员分级授权、专人专机负责制、信息化管理、PDCA循环法持续改进等管理措施为精准病理学诊断打好基础,大大提高了诊断准确率和及时率,为临床病理学诊断提供了可靠的技术支撑。