Engineered human angiogenin mutations in the placental ribonuclease inhibitor complex for anticancer therapy: Insights from enhanced sampling simulations

Targeted human cytolytic fusion proteins (hCFPs) represent a new generation of immunotoxins (ITs) for the specific targeting and elimination of malignant cell populations. Unlike conventional ITs, hCFPs comprise a human/humanized target cell‐specific binding moiety (e.g., an antibody or a fragment thereof) fused to a human proapoptotic protein as the cytotoxic domain (effector domain). Therefore, hCFPs are humanized ITs expected to have low immunogenicity. This reduces side effects and allows long‐term application. The human ribonuclease angiogenin (Ang) has been shown to be a promising effector domain candidate. However, the application of Ang‐based hCFPs is largely hampered by the intracellular placental ribonuclease inhibitor (RNH1). It rapidly binds and inactivates Ang. Mutations altering Ang's affinity for RNH1 modulate the cytotoxicity of Ang‐based hCFPs. Here we perform in total 2.7 µs replica‐exchange molecular dynamics simulations to investigate some of these mutations—G85R/G86R (GGRR_mut), Q117G (QG_mut), and G85R/G86R/Q117G (GGRR/QG_mut). GGRR_mut turns out to perturb greatly the overall Ang‐RNH1 interactions, whereas QG_mut optimizes them. Combining QG_mut with GGRR_mut compensates the effects of the latter. Our results explain the in vitro finding that, while Ang GGRR_mut‐based hCFPs resist RNH1 inhibition remarkably, Ang WT‐ and Ang QG_mut‐based ones are similarly sensitive to RNH1 inhibition, whereas Ang GGRR/QG_mut‐based ones are only slightly resistant. This work may help design novel Ang mutants with reduced affinity for RNH1 and improved cytotoxicity.     

Rational design of crystal contact‐free space in protein crystals for analyzing spatial distribution of motions within protein molecules

Contacts with neighboring molecules in protein crystals inevitably restrict the internal motions of intrinsically flexible proteins. The resultant clear electron densities permit model building, as crystallographic snapshot structures. Although these still images are informative, they could provide biased pictures of the protein motions. If the mobile parts are located at a site lacking direct contacts in rationally designed crystals, then the amplitude of the movements can be experimentally analyzed. We propose a fusion protein method, to create crystal contact‐free space (CCFS) in protein crystals and to place the mobile parts in the CCFS. Conventional model building fails when large amplitude motions exist. In this study, the mobile parts appear as smeared electron densities in the CCFS, by suitable processing of the X‐ray diffraction data. We applied the CCFS method to a highly mobile presequence peptide bound to the mitochondrial import receptor, Tom20, and a catalytically relevant flexible segment in the oligosaccharyltransferase, AglB. These two examples demonstrated the general applicability of the CCFS method to the analysis of the spatial distribution of motions within protein molecules.     

Steric hindrance of SNARE transmembrane domain organization impairs the hemifusion‐to‐fusion transition

SNAREs fuse membranes in several steps. Trans‐SNARE complexes juxtapose membranes, induce hemifused stalk structures, and open the fusion pore. A recent penetration model of fusion proposed that SNAREs force the hydrophilic C‐termini of their transmembrane domains through the hydrophobic core of the membrane(s). In contrast, the indentation model suggests that the C‐termini open the pore by locally compressing and deforming the stalk. Here we test these models in the context of yeast vacuole fusion. Addition of small hydrophilic tags renders bilayer penetration by the C‐termini energetically unlikely. It preserves fusion activity, however, arguing against the penetration model. Addition of large protein tags to the C‐termini permits SNARE activation, trans‐SNARE pairing, and hemifusion but abolishes pore opening. Fusion proceeds if the tags are detached from the membrane by a hydrophilic spacer or if only one side of the trans‐SNARE complex carries a protein tag. Thus, both sides of a trans‐SNARE complex can drive pore opening. Our results are consistent with an indentation model in which multiple SNARE C‐termini cooperate in opening the fusion pore by locally deforming the inner leaflets.     

A new procedure for the cloning,expression and purification of the β-carbonic anhydrase from the pathogenic yeast Malassezia globosa,an anti-dandruff drug target

Malassezia yeasts are almost exclusively the single eukaryotic members of the fungal flora of the skin. Malassezia globosa and Malassezia restricta are found on the skin of practically all humans. Malassezia globosa is highly implicated in the pathogenesis of dandruff and its genome encodes for only one carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the β-class (MgCA). It has been indeed demonstrated that in many pathogenic microorganisms, CAs are essential for their life cycle and their inhibition can lead to growth impairment and defects. In the previous work, the recombinant MgCA was investigated for its inhibition profile with sulfonamides, which in models of dandruff infection were able to protect animals from the fungal infection, allowing us to propose this enzyme as a new antidandruff target. MgCA was cloned as GST-fusion protein, but the yield was rather low and the protein was often found in inclusion bodies. Here, we propose an alternative procedure consisting in cloning the recombinant MgCA as His-Tag fusion protein. This procedure resulted in a good method to express and purify the active recombinant MgCA, and the protein recovery was better with respect to that used for preparing MG-CA (β-CA cloned as GST-fusion protein).     

Comparative role of disc degeneration and ligament failure on functional mechanics of the lumbar spine

Understanding spinal kinematics is essential for distinguishing between pathological conditions of spine disorders, which ultimately lead to low back pain. It is of high importance to understand how changes in mechanical properties affect the response of the lumbar spine, specifically in an effort to differentiate those associated with disc degeneration from ligamentous changes, allowing for more precise treatment strategies. To do this, the goals of this study were twofold: (1) develop and validate a finite element (FE) model of the lumbar spine and (2) systematically alter the properties of the intervertebral disc and ligaments to define respective roles in functional mechanics. A three-dimensional non-linear FE model of the lumbar spine (L3-sacrum) was developed and validated for pure moment bending. Disc degeneration and sequential ligament failure were modelled. Intersegmental range of motion (ROM) and bending stiffness were measured. The prediction of the FE model to moment loading in all three planes of bending showed very good agreement, where global and intersegmental ROM and bending stiffness of the model fell within one standard deviation of the in vitro results. Degeneration decreased ROM for all directions. Stiffness increased for all directions except axial rotation, where it initially increased then decreased for moderate and severe degeneration, respectively. Incremental ligament failure produced increased ROM and decreased stiffness. This effect was much more pronounced for all directions except lateral bending, which is minimally impacted by ligaments. These results indicate that lateral bending may be more apt to detect the subtle changes associated with degeneration, without being masked by associated changes of surrounding stabilizing structures.     

Ultrasonic Welding of Thermoplastic Composite Coupons for Mechanical Characterization of Welded Joints through Single Lap Shear Testing

This paper presents a novel straightforward method for ultrasonic welding of thermoplastic-composite coupons in optimum processing conditions. The ultrasonic welding process described in this paper is based on three main pillars. Firstly, flat energy directors are used for preferential heat generation at the joining interface during the welding process. A flat energy director is a neat thermoplastic resin film that is placed between the parts to be joined prior to the welding process and heats up preferentially owing to its lower compressive stiffness relative to the composite substrates. Consequently, flat energy directors provide a simple solution that does not require molding of resin protrusions on the surfaces of the composite substrates, as opposed to ultrasonic welding of unreinforced plastics. Secondly, the process data provided by the ultrasonic welder is used to rapidly define the optimum welding parameters for any thermoplastic composite material combination. Thirdly, displacement control is used in the welding process to ensure consistent quality of the welded joints. According to this method, thermoplastic-composite flat coupons are individually welded in a single lap configuration. Mechanical testing of the welded coupons allows determining the apparent lap shear strength of the joints, which is one of the properties most commonly used to quantify the strength of thermoplastic composite welded joints.     

右美托咪定预防全麻下后路减压椎间植骨融合术后寒战的临床研究

目的:观察右美托咪定对全麻下后路减压椎间植骨融合术(PLIF)后寒战的预防效果。方法:选择行全身麻醉的PLIF手术患者80例,并将其随机分为高剂量右美托咪定组(HD组)、中剂量右美托咪定组(MD组)、低剂量右美托咪定组(LD组)和对照组(C组),每组20例患者。HD组、MD组及LD组在麻醉诱导后分别以0.8、0.5及0.2μg/kg/h静脉泵注右美托咪定至手术结束前40 min;C组泵注生理盐水。监测并记录患者麻醉前的基础体温值及BIS值,送入恢复室即刻、20 min、40 min、60 min的Ramsay镇静评分值、体温值;记录各组手术时长、拔管时间、术中输液量及失血量以及心动过缓、恶心、呕吐等不良反应的发生情况和苏醒期各组寒战程度分级及发生情况。结果:各组患者手术结束时的体温均较基础体温明显降低(P0.05)。入恢复室后,HD组同一时段的Ramsay镇静评分显著高于其他三组(P0.01)。HD组的拔管时间明显延长(P0.01)。与C组比较,MD组及HD组术后心动过缓、口干的发生率明显升高(P0.05),而恶心、呕吐及呛咳的发生率明显降低(P0.05)。在恢复室观察期间,MD组及HD组寒战的发生率较C组明显降低(P0.01);MD组寒战发生率也明显低于LD组(P0.05)。结论:0.5μg/kg/h持续泵注右美托咪定可以有效预防全麻下PLIF手术术后寒战的发生。     

米曲霉原生质体的营养互补融合及二倍体的诱发分离

采用1％溶壁酶加1％蜗牛酶的混合液获得的原生质体,以30％聚乙二醇(MW=6,000)、0.01M CaCl_2、0.05M Gly做为融合剂,对米曲霉进行了原生质体的营养互补融合,融合频率为0.27—0.47％。自4个菌株的4对杂交组合中获得了异核体,并分离到97株绿色融合株。二倍体的孢子经PFA和UV诱发分离后,获得了二株生长速度快、蛋白酶活性高和产孢能力强的单倍体菌株。     

Uptake of protoplasts from the filamentous fungiAspergillus nidulans andFusarium oxysporum into protoplasts from celery (Apium graveolens L.)

Summary Protoplasts isolated from celery cell suspension cultures, were mixed with fungal protoplasts, from either the saprophytic speciesAspergillus nidulans or the pathogenic speciesFusarium oxysporum. The incubation of protoplast mixtures with PEG caused close adhesion between plant and fungal protoplasts. Subsequent dilution of PEG resulted in the uptake of protoplasts from either fungal species into the plant protoplast cytoplasm. A range of PEG concentrations, incubation times and dilution rates were tested to maximise adhesion and uptake frequencies. Identification of uptake was achieved either by fluorescent staining of nuclei or by electron-microscopy. A maximum of 10% celery protoplasts had taken upA. nidulans protoplasts after PEG treatment. Fungal protoplasts were taken up into celery protoplast cytoplasm by endocytosis, and were maintained within vesicles; two bounding membranes were observed by electron microscopy. Plant protoplast viability was determined during prolonged incubation following fungal protoplast uptake. The presence ofA. nidulans protoplasts tended to maintain celery protoplast viability and although some morphological disintegration occurred intact celery protoplasts remained for at least 92 h after uptake. The uptake ofF. oxysporum protoplasts markedly depressed celery protoplast viability after 24 h incubation and greater celery protoplast disintegration occurred.Abbreviations PEG Polyethylene glycol - DAPI 4,6-diaminido-2-phenylindole - 2,4-D 2,4-dichlorophenoxyacetic acid