Dynamic allostery governs cyclophilin A–HIV capsid interplay

Host factor protein Cyclophilin A (CypA) regulates HIV-1 viral infectivity through direct interactions with the viral capsid, by an unknown mechanism. CypA can either promote or inhibit viral infection, depending on host cell type and HIV-1 capsid (CA) protein sequence. We have examined the role of conformational dynamics on the nanosecond to millisecond timescale in HIV-1 CA assemblies in the escape from CypA dependence, by magic-angle spinning (MAS) NMR and molecular dynamics (MD). Through the analysis of backbone ¹H-¹⁵N and ¹H-¹³C dipolar tensors and peak intensities from 3D MAS NMR spectra of wild-type and the A92E and G94D CypA escape mutants, we demonstrate that assembled CA is dynamic, particularly in loop regions. The CypA loop in assembled wild-type CA from two strains exhibits unprecedented mobility on the nanosecond to microsecond timescales, and the experimental NMR dipolar order parameters are in quantitative agreement with those calculated from MD trajectories. Remarkably, the CypA loop dynamics of wild-type CA HXB2 assembly is significantly attenuated upon CypA binding, and the dynamics profiles of the A92E and G94D CypA escape mutants closely resemble that of wild-type CA assembly in complex with CypA. These results suggest that CypA loop dynamics is a determining factor in HIV-1''s escape from CypA dependence.Cyclophilin A (CypA), a peptidyl-prolyl isomerase, is a host factor critical in the regulation of the HIV-1 infection, involving a direct interaction with the capsid (CA) protein (1–3). The mechanism by which CypA modulates the viral infectivity is complex and poorly understood, being dependent on the CA protein primary sequence and the host cell type (4–6). For example, it is known that mutations in the CypA-binding loop of the CA protein dramatically reduce virus infectivity (7, 8). The A92E and G94D escape mutants bind CypA with similar affinity to wild-type CA, but exhibit only 10% of the activity of wild-type CA in the presence of CypA, and full infectivity can be restored if CypA is inhibited with cyclosporin A in the host cells (8), as shown schematically in SI Appendix, Fig. S1. Alas, the molecular mechanisms underlying CypA escape remain elusive, despite numerous virological, biochemical, and structural–biological studies.The present study investigates the internal conformational dynamics of a CA protein assembly. Although static structures of HIV-1 proteins and complexes with host factors provide important clues into their assembly architecture and conformational details of the interactions, structures alone are insufficient for understanding molecular mechanisms. It is well known that biological functions can be dynamically regulated, at multiple levels of organization, from internal dynamics of individual protein molecules (9) to entire cells. This dynamic regulation certainly also applies to HIV-1 because numerous dynamic processes are associated with HIV-1 assembly, disassembly, release, and maturation (10, 11). For example, we previously demonstrated that internal conformational dynamics of the CA protein and its structural plasticity determine its ability to assemble into pleiomorphic conical capsids (12, 13) (Fig. 1). We also uncovered that, in the HIV-1 CA-SP1 maturation intermediate, dynamic disorder in the SP1 peptide plays an important role in the final step of virus maturation, permitting condensation of CA into the cores of infectious virions (14).Open in a separate windowFig. 1.(A, Left) All-atom MD-derived model of mature HIV-1 capsid constructed on the basis of cryo-electron tomography (cryo-ET) and solution NMR studies (13). The capsid comprises 216 hexamers (orange) and 12 pentamers (blue) [Protein Data Bank (PDB) ID 3J3Y]. Structural organization of a hexamer of hexamers (HOH) building block is illustrated in the expansion. Color coded are individual hexameric units comprising the HOH building block. (A, Right) The 3D structure of CA monomer [HXB2 sequence polymorph [PDB file 3NTE (42)]. (B) Cosedimentation assay of CA with CypA illustrating the efficiency of cosedimentation for different CA/CypA molar ratios. S, supernatant; P, pellet. (C) Transmission electron microscopy (TEM) images of tubular assemblies of CA and CA/CypA. (C, Upper) CA NL4-3 (Left), CA NL4-3 A92E (Center), and CA NL4-3 G94D (Right). (C, Lower) HXB2 (Left) and CA HXB2/CypA (Right). (D) Expansions around the aliphatic region for 2D NCA and combined R2-driven (CORD) MAS NMR spectra for CA HXB2 (black) and CA HXB2/CypA (orange), illustrating the multiple chemical shift perturbations observed upon formation of the complex. These perturbations are mapped onto the structure of CA monomer (A) and are confined to flexible loops and residue variation sites. The spectra are recorded at 20.0 T and the MAS frequency of 14 kHz. (E) Expansions of glycine regions for 2D NCA MAS NMR spectra for (from left to right): HXB2, HXB2/CypA, NL4-3, NL4-3 A92E, and NL4-3 G94D. Dashed lines indicate the G89 cross-peaks associated with cis- and trans-P90.In this study, we examined the residue-specific mobility of CA protein from HXB2 and NL4-3 sequence polymorphs (SI Appendix, Fig. S2) in tubular assemblies on the nanoseconds to milliseconds timescales. In particular, we compared wild-type and A92E and G94D escape mutants of the NL4-3 strain as well as wild-type HXB2 CA alone and in complex with CypA. As discussed previously (14, 15), tubular assemblies recapitulate the hexameric lattice, the predominant symmetry arrangement of the conical HIV-1 capsid core, illustrated in Fig. 1A. Dipolar tensors and resonance intensities extracted from a series of 2D and 3D homonuclear and heteronuclear magic-angle spinning (MAS) NMR experiments revealed that certain regions in both HXB2 and NL4-3 wild-type CA are unusually dynamic on all timescales. These motions are significantly attenuated upon CypA binding. Most remarkably, the dynamic profiles of the A92E and G94D escape mutants closely resemble that of CA when bound by CypA. To gain further understanding of the sequence-dependent dynamics profiles of CA assemblies, we performed extensive molecular dynamics (MD) simulations. The motionally averaged dipolar tensors extracted from the MD trajectories are in remarkable quantitative agreement with the NMR results. Together, our results suggest that changes in the sequence-dependent conformational dynamics may be a key determinant in the escape mechanism of HIV-1 CA capsid mutants from CypA dependence.     

细菌脂多糖对选择性激光熔化技术制作的钴铬钼合金腐蚀行为的影响

目的 研究不同浓度细菌脂多糖（Lipopolysaccharides,LPS）对选择性激光熔化技术（Selective laser melting,SLM）制作的钴铬钼合金腐蚀行为的影响。方法 分别以SLM法及铸造法各制作12个钴铬钼合金试件,根据在腐蚀电解液中LPS浓度（0、0.15、15、150 μg/mL）不同分为4组,每组3个试件。采用电化学阻抗谱、动电位极化测试法分析2组合金试件在不同浓度脂多糖（LPS）中的腐蚀行为。结果 在单纯人工唾液中,SLM合金和铸造合金的腐蚀参数无统计学上的差异（P> 0.05）。当LPS浓度为150 μg/mL时,SLM合金和铸造合金的电阻（R_p）均显著下降且自腐蚀电流密度（I_corr）显著增大,但SLM合金的R_p值大于铸造合金且I_corr值较小（P< 0.05）。结论 LPS对SLM法和铸造法制作的钴铬钼合金的腐蚀行为均有不利影响,但对SLM法制作的合金的腐蚀影响小于铸造法制得的合金。     

Vitritis and retinal vasculitis caused by pseudorabies virus

Pseudorabies virus (PRV) is a herpesvirus of swine. PRV is also called suid herpesvirus 1 and is a member of the Alphaherpesvirinae subfamily within the family Herpesviridae. The number of PRV cases worldwide is small, but in susceptible individuals, infection with this virus has a poor prognosis. Therefore, it is urgent to improve our understanding of this disease in clinical practice to avoid misdiagnosis and to identify optimal treatments. We report a patient with PRV infection who was admitted to hospital with viral encephalitis and subsequently developed intraocular infection. Because to the lack of relevant clinical experience in the treatment of this disease, we carried out experimental treatment with good therapeutic effect. This case provides a basis for clinical diagnosis and treatment of patients with PRV.     

吸烟对男性肺癌患者组织学分型的影响及其趋势分析

背景与目的 已有的研究发现我国肺癌患者的组织学亚型分布有变化.本研究旨在探讨吸烟对我国男性肺癌患者组织学分型的影响及变化趋势.方法 收集和整理2000年-2012年中国医学科学院肿瘤医院诊治的男性肺癌患者人口学、吸烟史、组织学等信息,用年度百分比变化(annual percentage change,APC)进行趋势检验.结果 入选肺癌14,106例,其中吸烟11,750例,不吸烟2,356例.吸烟患者中,鳞癌的比例为39.38%,腺癌29.85%;2000年-2012年,鳞癌比例从44.19%下降至35.50%(APC=-1.9%,P<0.001);腺癌从15.25%上升至41.85%(APC=6.8%,P<0.001);腺鳞癌从4.13%降低至0.72%(APC=-14.9%,P<0.001).不吸烟患者中,腺癌53.86%,鳞癌16.64%;腺癌从38.03%上升至67.83%(APC=4.3%,P<0.001);大细胞癌和腺鳞癌呈波动变化.结论 肺腺癌在男性吸烟和非吸烟肺癌患者中的比例均显著上升,其非吸烟因素暴露与肺癌的关系应进一步深入研究.     

miR-147a靶向MCM3抑制脉络膜黑色素瘤细胞增殖、迁移和侵袭

目的:研究miR-147a对脉络膜黑色素瘤细胞增殖、迁移和侵袭的影响,并探究其作用机制.方法:采用脂质体转染人脉络膜黑色素瘤MUM-2B细胞,设置miR-NC、miR-147a mimics、inhibitor-NC、miR-147a inhibitor四组.采用EDU染色法检测细胞增殖;Transwell实验检测细胞...     

371 例肿瘤姑息治疗多学科会诊临床分析*

目的：分析天津医科大学肿瘤医院肿瘤姑息治疗多学科专家协作组（Multidisciplinary team ,MDT ）的工作概况,评价MDT 模式在肿瘤姑息治疗领域的作用。方法：回顾性分析2014年1 月1 日至2015年3 月31日参加本院肿瘤姑息治疗MDT 会诊的371 例患者的临床资料,总结其基本特征、MDT 决策执行情况,并对部分姑息治疗方案进行疗效评价。结果：全组患者401 例次共获得318（79%）项治疗建议,其中293 项为姑息治疗建议。MDT 为119 例疼痛患者提供了合理化镇痛方案,对95例患者提出营养治疗意见,对36例患者采取了抗焦虑抑郁药物治疗。有效随访到的374 项MDT 决策,全部执行者273 项（73%）,部分执行者49项（13%）,总体执行率86% 。执行MDT 决策的患者,在疼痛控制、营养状况改善及与抑郁相关的躯体症状缓解方面均优于未执行者（P < 0.001,P < 0.001 及P < 0.01）。 结论：肿瘤姑息治疗多学科会诊能为患者提供规范的个体化综合治疗建议,执行MDT 决策有助于改善癌症患者疼痛、营养障碍及焦虑抑郁等症状,值得进一步完善和推广。     

顺铂脂质体腹腔注射对S180荷瘤小鼠的治疗作用

目的 探讨顺铂脂质体腹腔注射对S180荷瘤小鼠的抗肿瘤作用.方法 采用昆明种小鼠腹腔内注射S180细胞形成腹水瘤模型,40只小鼠肿瘤种植24 h后随机分为4组,分别为生理盐水组、空白脂质体组、顺铂注射液组、顺铂脂质体组,分别给予生理盐水、空白脂质体、顺铂注射液和顺铂脂质体腹腔注射,共给药4次,给药时间间隔72 h.结果...     

Clinical significance of circulating tumor cells in predicting disease progression and chemotherapy resistance in patients with gestational choriocarcinoma

Gestational choriocarcinoma (GC) is a highly aggressive tumor. In our study, we systematically investigated EpCAM/CD147 expression characteristics in patients with GC and assessed the role of circulating tumor cells (CTCs) in predicting chemotherapy response and disease progression. GC tissues were positive for either epithelial cellular adhesion molecule (EpCAM) or CD147, and all samples exhibited strong human chorionic gonadotropin (HCG) expression. Among all the recruited patients (n = 115), 103 had at least 1 CTC in a 7.5-mL peripheral blood sample, and the percentage of patients with ≥4 CTCs in a particular FIGO stage group increased with a higher FIGO stage (p < 0.001). Furthermore, the pretreatment CTC count was related to tumor size (r = 0.225, p = 0.015) and the number of metastases (r = 0.603, p < 0.001). A progression analysis showed that among the 115 included patients who qualified for further examination, 52 of the 64 patients defined as progressive had ≥4 pretreatment CTCs, while only 7 of the 51 non-progressive patients had ≥4 pretreatment CTCs (p < 0.001). In multivariate analysis, CTCs (≥4) remained the strongest predictor of PFS when other prognostic markers, FIGO score and FIGO stage were included. Moreover, based on the chemotherapy response, patients with ≥4 CTCs were more likely to be resistant to chemotherapy than those with <4 CTCs (P < 0.001). These findings demonstrates the feasibility of CTC detection in cases of GC by adopting EpCAM/CD147 antibodies together as capturing antibodies. The CTC count is a promising indicator in the evaluation of biological activities and the chemotherapy response in GC patients.     

基于中西医临床病证特点的系统性红斑狼疮动物模型研究评述

基于系统性红斑狼疮（SLE）的中西医临床病证特点,参考国内外SLE动物模型研究文献,依据临床诊断标准,对现有模型的临床符合度进行评价,经对比分析发现,自发小鼠模型中的MRL/lpr小鼠模型与诱导模型中的姥鲛烷诱导模型中西医临床病证吻合度最高。但目前SLE动物模型多按照西医病因特点建模,缺乏中医病证结合模型,中医符合度低,因此在现有研究基础上增加中医干扰因素,彰显中医辨证优势,加强SLE中医辨证相关研究是今后努力的方向。