CD44基因多态性与宫颈癌及非小细胞肺癌的相关性

背景与目的：CD44分子是众多肿瘤细胞的标志分子，其表达水平与肿瘤细胞的恶性程度有关。该研究探讨CD44基因中的单核苷酸多态性（single nucleotide polymorphism，SNP）位点与云南汉族人群宫颈癌和非小细胞肺癌（nonsmall cell lung cancer，NSCLC）易感性的相关性。方法：选取了CD44基因中的两个SNP位点rs13347和rs8193，采用TagMan基因分型的方法，分析这两个多态性位点在497例宫颈癌患者和500例健康对照个体以及483例NSCLC患者和471例健康对照个体中的分布特征，并分析CD44基因中的多态性位点与云南汉族人群宫颈癌和NSCLC的相关性。结果：rs13347和rs8193位点等位基因和基因型在宫颈癌组和对照组中的分布频率的差异无统计学意义（P>0.05）。而在NSCLC组和对照组的比较中：rs13347和rs8193位点等位基因在NSCLC组和对照组中的分布频率的差异有统计学意义（P=0.020和P=0.004）；这两个位点基因型在NSCLC组和对照组中分布频率的差异有统计学意义（P=0.027和P=0.020）；其中rs13347位点等位基因C在NSCLC组中的分布频率显著高于对照组，可能是NSCLC发生的风险因素（OR=1.250，95% CI：1.035～1.509），rs8193位点等位基因C在对照组中的分布频率显著高于NSCLC组，可能是NSCLC发生的保护性因素（OR=0.768，95%CI：0.641～0.921）。单倍型分析结果显示，rs13347C-rs8193T和rs13347T-rs8193C在NSCLC组和对照组中的分布频率差异有统计学意义（P=0.003和0.022）；该结果说明单倍型rs13347C-rs8193T可能是云南汉族人群NSCLC发生的风险性因素（OR=1.316，95%CI：1.096～1.579）。结论：CD44基因中的两个SNP位点rs13347和rs8193可能与云南汉族人群宫颈癌发病风险无关，而可能与云南汉族人群NSCLC具有相关性。     

T1~T2期乳腺癌保乳术后化疗后程同步大分割放疗前瞻性Ⅰ期临床研究

目的 前瞻性评估T1~T2期乳腺癌保乳术后化疗后程大分割放疗的不良反应和耐受性，以及在缩短治疗时间、减轻患者经济负担等方面的价值。方法 共入组20例T1~T2期乳腺癌保乳术后患者，所有患者于末次多西他赛化疗前开始大分割放疗。观察急性放射反应、治疗完成率及无病生存率、住院时间及住院费用等。结果 治疗完成率100%。主要不良反应为血液学毒性（白细胞减少）及皮肤反应，患者均可耐受。中位随访时间为30.1个月，随访率100%。美容效果良好率100%。平均总治疗时间为4周，总住院治疗费用节省约1万元。21个月无病生存率为100%。结论 T1~T2期乳腺癌保乳术后可耐受同步大分割放化疗，局部控制好，美容效果佳，且具有较高的卫生经济学价值。     

清前方对实验性慢性非细菌性前列腺炎的作用

目的 观察清前方对大鼠慢性非细菌性前列腺炎的作用。方法 采用向大鼠前列腺内注入1％角叉菜胶生理盐水的方法，建立实验性非细菌性前列腺炎模型，并将实验大鼠随机分为4组，分别给予相应剂量的蒸馏水（A组和B组）、清前方（C组）或舍尼通（D组）混悬液，观察清前方对该炎症模型大鼠体重增长变化、前列腺脏／体比值及病理学改变的影响。结果 清前方（2．5g／kg·d）组炎症模型动物体重增长回升，前列腺脏／体比值下降，前列腺平滑肌增殖受到抑制，局部病理性瘀血状态得以改善。结论 清前方可望用于治疗慢性非细菌性前列腺炎。     

丹参酮ⅡA通过抑制HDAC3影响巨噬细胞极化的作用研究

目的探讨丹参酮ⅡA(TanⅡA)通过抑制组蛋白去乙酰化酶3(HDAC3)影响巨噬细胞极化的作用。方法应用中药系统药理学数据库与分析平台(TCMSP)筛选TanⅡA药效靶点和动脉粥样硬化作用靶点,并将二者的交集基因进行KEGG通路分析,采用Cytoscape 3.7.1软件对主要交集基因-信号通路进行可视化分析。选用THP-1单核细胞株经佛波醇12-十四酸酯13-乙酸酯(PMA)诱导为贴壁的巨噬细胞后,分为M0组、氧化型低密度脂蛋白(ox-LDL)组、TanⅡA+ox-LDL组及ox-LDL+HDAC3 siRNA组。采用流式细胞术、免疫荧光实验及qRT-PCR技术检测TanⅡA对ox-LDL诱导的巨噬细胞极化方向的改变及HDAC3 mRNA表达水平的变化。结果生物信息学分析得到TanⅡA药效靶点和动脉粥样硬化作用靶点有23个交集基因,选取排序靠前的20条信号通路进行可视化分析,发现主要富集于动脉粥样硬化、流体剪切力和TNF信号通路等。其中,JUN、FOS、RELA、NFKBIA四个交集基因参与巨噬细胞极化的信号通路调控。流式细胞术、免疫荧光实验及qRT-PCR结果显示ox-LDL可诱导M0巨噬细胞CCR7和CCL2表达显著升高;与ox-LDL组相比,TanⅡA预处理的M0巨噬细胞经ox-LDL诱导后HDAC3、CCR7及CCL2 mRNA表达均下降,和ox-LDL+HDAC3 siRNA组结果一致。结论 TanⅡA能有效地阻止ox-LDL诱导巨噬细胞往M1方向极化,其机制可能是通过调控巨噬细胞HDAC3表达介导的。     

The effects of Sarconesiopsis magellanica larvae (Diptera: Calliphoridae) excretions and secretions on fibroblasts

Sarconesiopsis magellanica is a necrophagous blowfly which is relevant in both forensic and medical sciences. Previous studies regarding this species have led to understanding life-cycle, population and reproduction parameters, as well as identifying and characterising proteolytic enzymes derived from larval excretions and secretions (ES). As other studies have shown that ES proteolytic activity plays a significant role in wound healing and fibroblasts play a relevant role in granulation tissue formation during such healing, the present study was aimed at analysing the biological effect of S. magellanica larval ES on fibroblasts. ES were obtained from third-instar larvae and added to fibroblast cells at three concentrations (10, 5 and 1 μg/mL) to evaluate their behaviour. MTT assays were used for analysing cell proliferation and viability, whilst cell adhesion was measured by optical density with 10% SDS. Fibroblast migration and morphology was recorded by microscopic observation. ES did not affect fibroblast viability and induced an increase in cell proliferation; cell adhesion became reduced, whilst cell migration through extracellular matrix increased. ES also induced a decreased cell surface and morphological alterations. Changes in all the above-mentioned parameters were reduced when ES were incubated at 60 °C, probably due to protease denaturation. These results suggested that the proteases contained in S. magellanica larval ES contributed towards granulation tissue formation, increased cell migration and promoted cell proliferation. All these data support carrying out further experiments aimed at validating S. magellanica usefulness in larval therapy.     

Molecular dynamics simulations of the mechanical behavior of alumina coated aluminum nanowires under tension and compression

For materials with high oxygen affinity, oxide layers will significantly change the material properties. This is of particular importance for aluminum nanowires which have many applications because of their ultrahigh strengths. Recent studies show that thin amorphous oxide shell layers on aluminum surfaces significantly change the responses of the material. However, the relations between the thickness of the oxidized layer, the strain rate and the mechanical response of nanowires to compression and tension have not been investigated intensively. In this study, we use a ReaxFF potential to analyze the influences of oxide shell layers on the material responses of the nanowires under uniaxial tension and compression at different strain rates. The Al–O interface leads to an increased defect nucleation rate at the oxide interface preventing localized deformation. During tension, we observe a reorganization of the structure of the oxide layer leading to bond healing and preventing fracture. While ductility is increasing with coating thickness during tension, the thickness of the coating is less decisive during compression.

Alumina coatings increase the ductility of aluminum nanowires by reorganization of the Al–O layer and stabilization of bonds.     

Toughening of POSS–MPS composites with low dielectric constant prepared with structure controllable micro/mesoporous nanoparticles

In this work, we developed a modified calcination and extraction method to obtain controllable micro/mesoporous nanoparticle samples POSS–MPS, which were synthesized through glycidyl polyhedral oligomeric silsesquioxane (G-POSS) grafting with aminopropyl-functionalized mesoporous silica (AP-MPS). The POSS–MPS was introduced into the cyanate ester (CE) matrix to optimize the dielectric properties and enhance the toughness of the POSS–MPS/CE nanocomposite. The structure of the hybrid was characterized by FTIR and SEM. The dispersion properties, mechanical properties, dielectric properties and thermal performance were also studied. The results showed that both the C-POSS–MPS and E-POSS–MPS uniformly distribute in the CE matrix with the content of 0.5–4 wt%. The impact strength increased 52% and 60% separately with 2 wt% C-POSS–MPS and E-POSS–MPS addition respectively. The introduction of E-POSS–MPS particles can significantly decrease the dielectric loss value of the POSS–MPS/CE composites to 0.00498, which is of potential in wave transparent composites and structures.

A promising method to improve the performance of CE composites via combining advantages of POSS and MPS.     

芎麻滴丸对血管性认知障碍大鼠社交行为的影响

目的 观察双侧颈总动脉结扎致血管性认知障碍大鼠社交行为的变化,探讨芎麻滴丸对其干预作用。方法 采用永久性结扎双侧颈总动脉法制备血管性认知障碍模型。实验大鼠随机分为假手术组（生理盐水10 mL·kg^-1）,模型组（生理盐水10 mL·kg^-1）,芎麻滴丸高剂量组（生药1.5 g·kg^-1）,芎麻滴丸低剂量组（生药0.75 g·kg^-1）。模型制备第28,42天采用Morris水迷宫对大鼠的学习记忆能力进行测试;模型制备第7,14,28,42天用社交行为仪检测大鼠社交能力的变化。结果 模型制备第7,14天大鼠社交行为无明显变化,模型制备第28天和第42天,模型组大鼠持续接触时间减少（P<0.05）;模型制备第42天,模型组大鼠接触次数减少,逃避潜伏期显著增加,穿越平台次数显著减少（P<0.01）。与模型组大鼠相比,模型制备第28天,芎麻滴丸高剂量组大鼠接触次数显著增加（P<0.05）;芎麻滴丸高、低剂量组大鼠持续接触时间显著增加（P<0.05或<0.01）;模型制备第42天,芎麻滴丸低剂量组大鼠接触次数和持续接触时间增加（P<0.05）,芎麻滴丸高、低剂量组大鼠逃避潜伏期显著减少（P<0.01）。结论 模型制备第42天大鼠出现学习记忆障碍;模型组大鼠第28天大鼠开始出现社交行为障碍,经芎麻滴丸干预后,接触时间和接触次数增加,即可认为其有效缓解由双侧颈总动脉结扎造成的社交能力障碍,且低剂量效果优于高剂量。     

Dual-field-of-view high-spectral-resolution lidar: Simultaneous profiling of aerosol and water cloud to study aerosol–cloud interaction

Aerosol–cloud interaction (ACI) is complex and difficult to be well represented in current climate models. Progress on understanding ACI processes, such as the influence of aerosols on water cloud droplet formation, is hampered by inadequate observational capability. Hitherto, high-resolution and simultaneous observations of diurnal aerosol loading and cloud microphysical properties are challenging for current remote-sensing techniques. To overcome this conundrum, we introduce the dual-field-of-view (FOV) high-spectral-resolution lidar (HSRL) for simultaneously profiling aerosol and water cloud properties, especially water cloud microphysical properties. Continuous observations of aerosols and clouds using this instrument, verified by the Monte Carlo simulation and coincident observations of other techniques, were conducted to investigate the interactions between aerosol loading and water cloud microphysical properties. A case study over Beijing highlights the scientific potential of dual-FOV HSRL to become a significant contributor to the ACI investigations. The observed water cloud profiles identify that due to air entrainment its vertical structure is not perfectly adiabatic, as assumed by many current retrieval methods. Our ACI analysis shows increased aerosol loading led to increased droplet number concentration and decreased droplet effective radius—consistent with expectations—but had no discernible increase on liquid water path. This finding supports the hypothesis that aerosol-induced cloud water increase caused by suppressed rain formation can be canceled out by enhanced evaporation. Thus, these observations obtained from the dual-FOV HSRL constitute substantial and significant additions to understanding ACI process. This technique is expected to represent a significant step forward in characterizing ACI.

Aerosol–cloud interaction (ACI) is a crucial aspect of atmospheric research and one of the primary sources of uncertainties in climate predictions (1–3). To assess the credibility of climate projections, it is imperative to improve our understanding of how aerosols interact with clouds (4–6). It has been well known that aerosols can serve as cloud condensation nuclei (CCN) to form cloud droplets, which can further influence the initiation of precipitation (7). However, quantifying the impact of natural and anthropogenic aerosols on the growth and the evolution of water clouds is still challenging (8, 9). The short lifetime, high temporal variability, and complex vertical structure of water cloud layers lead to a major difficulty for ACI studies (3, 10, 11). Despite the advances in the characterization of ACI by ground-based measurements (12–21), satellite retrieved products (22–25), and airborne in situ measurements (26–28), uncertainties remain in the effects of the aerosols on the water cloud properties. The reason for this gap in our knowledge is closely linked to the inadequate observations of the water cloud microphysical properties under various aerosol conditions (3). Current satellites can estimate cloud properties but not the typical aerosol nucleation region beneath clouds (22–25). Moreover, they also bring challenges for ACI studies that the typical revisit time of satellite-based sensors is much longer than the temporal scales of cloud variability (29, 30). Quintessential ground-based remote-sensing techniques for retrieving cloud properties, such as the cloud radar and the microwave radiometer, cannot provide simultaneous aerosol observations for ACI studies. Therefore, ground-based measurements commonly combine those with other remote-sensing or in situ aircraft instruments for characterizing aerosol loading beneath clouds (12–20). However, given the high variability of clouds, differences in perspective or mismatched sampling in space and time would raise uncertainty and bias in the characterization of ACI (15).Lidar, a powerful tool for profiling optical properties of aerosols and clouds, has been widely used in atmospheric studies (31–33). Yet, further progress with lidar-based techniques for ACI studies is hampered by limited observations of the water cloud microphysical properties, mainly due to the difficulties of quantifying the multiple scattering within water clouds (34). The multiple scattering has a significant impact on the water cloud observations of the extinction as well as the depolarization ratio, which is related to the receiver field of view (FOV). In brief, a retrieval of water cloud microphysical properties for lidar-based techniques relies on utilizing different receiver FOVs to provide the necessary observations for characterizing the multiple-scattering effect caused by the water droplets (34, 35). The first multiple-FOV lidars were aimed at investigating the multiple-scattering effect and measured Mie scattering by water droplets (36). However, a complicated behavior of the Mie phase function makes the quantifying of the multiple scattering become an arduous task. It naturally leads to the use of Raman scattering of atmospheric nitrogen, which has an isotropic phase function practically in the backward direction, to allow developing a feasible algorithm for the retrieval of water cloud microphysical properties (35). Moreover, the dual-FOV Raman lidar technique for profiling cloud properties has been experimentally demonstrated (37). With this technique in conjunction with an incoherent Doppler lidar, the ACI findings have been obtained with an ACI index versus vertical air motion (21, 38). However, nitrogen Raman signals are so weak that the observations are usually restricted to nighttime hours, and the signal has to be averaged over tens of minutes to deliver reliable lidar products, while the typical temporal scale of cloud variability is much shorter than that (29). Recently, a dual-FOV polarization lidar technique was reported, which continued and further developed the concept of the dual-FOV Mie lidar (39). However, to assess ACI this method requires a priori assumptions about the lidar ratio and subadiabatic cloud conditions. The impact of the a priori assumptions on the aerosol and cloud retrievals has been widely discussed (10, 40, 41). In general, all existing multiple-FOV lidar-based techniques have their advantages and also limitations.To overcome this conundrum, a dual-FOV high-spectral-resolution lidar (HSRL) technique for profiling aerosol and cloud properties simultaneously is introduced here. It provides lateral observations of aerosols and clouds with high vertical and temporal resolutions during daytime and nighttime. Neither assumptions on thermodynamic conditions nor lidar ratio are required. This work benefited from the range-resolved observations of water clouds with high resolution, revealing that the observed profiles of low-level water cloud microphysical properties are not perfectly adiabatic as assumed by many current retrievals (42–46). Furthermore, the ACI analysis supports the hypothesis that aerosol-induced water decrease by enhanced evaporation can cancel out the increase caused by suppressed rain formation (6, 47), while most current global general circulation models (GCMs) suggest that increased aerosol loading typically causes increased cloud water content (19, 48). Thus, these observations obtained from the dual-FOV HSRL can constitute a substantial and significant addition to our understanding of ACI studies. We believe that this versatile system will not only benefit the quality monitoring of aerosol and cloud properties but also serve as a powerful tool for ACI studies.     

Development of an MRI contrast agent for both detection and inhibition of the amyloid-β fibrillation process

A curcumin derivative conjugated with Gd-DO3A (Gd-DO3A-Comp.B) was synthesised as an MRI contrast agent for detecting the amyloid-β (Aβ) fibrillation process. Gd-DO3A-Comp.B inhibited Aβ aggregation significantly and detected the fibril growth at 20 μM of Aβ with 10 μM of probe concentration by T₁-weighted MR imaging.

A curcumin derivative conjugated with Gd-DO3A (Gd-DO3A-Comp.B) was developed to significantly inhibit the amyloid-β (Aβ) aggregation and detect the fibril growth by T₁-weighted MR imaging.

A significant increase of Alzheimer''s disease (AD) patients urges the development of therapeutic and diagnostic technology.¹ As with the therapeutic development, diagnostic technology also faces several obstacles. To date, the definite diagnosis of AD relies on the histopathological data of post-mortem.^2,3 The non-invasive imaging technology targeting AD biomarkers such as amyloid β (Aβ) could provide phenotypical diagnostics, although the development of Aβ probes still remains challenging. Several contrast agents for single photon emission computed tomography (SPECT) and positron emission tomography (PET) such as Florbetapir-¹⁸F and Pittsburgh compound-B ([¹¹C]PiB) were developed as efficient tracers in mild cognitive impairment patients.^4,5 However, PET- and SPECT-based diagnostics require injection of radioactive probes, which cannot be measured frequently due to radiation exposure and limited availability of facilities. They also provide limited information on the anatomic profile of biomarkers due to their low spatial resolution and imprecise microscopic localization.⁶ In contrast, magnetic resonance imaging (MRI) contrast agents could quantify the Aβ accumulation in the anatomic brain image.⁷Several reported MRI contrast agents using gadolinium (Gd) complexes demonstrate potential use of Aβ detection. A clinically approved contrast agent, Gd(iii) diethylenetriaminepentaacetic acid (Gd-DTPA) complex accumulates in brain after opening the blood–brain barrier (BBB) by using mannitol and detects Aβ deposits in the mice AD-model.⁸ To improve the selectivity, Gd complexes were conjugated with compounds binding to Aβ such as Pittsburgh compound B (Gd-DO3A-PiB) which also serves as an approach for increasing MRI sensitivity.^9,10 An α,β-unsaturated ketone compound curcumin has been widely reported as an Aβ probe due to its ability to bind the hydrophobic site of Aβ.^11,12 Allen et al. firstly reported the direct conjugation of curcumin with Gd-DTPA which binds to Aβ with four times higher relaxivity than free Gd-DTPA.¹³ Furthermore, a polymalic acid-based nanoparticle covalently linked with curcumin and Gd-DOTA could also detect Aβ in human brain specimen by MRI.¹⁴ These previous studies demonstrate that the curcumin structure has significant potential for the development of MRI contrast agents for AD diagnosis.Previously, we reported a curcumin derivative, compound B, possesses 100-times stronger inhibitory activity of Aβ aggregation than curcumin on the basis of thioflavin T (ThT) competitive binding assay.^15,16 According to this result, we designed curcumin-based Gd probes for the detection and inhibition of Aβ (Fig. 1A–C). We hypothesized that these probes could accelerate proton longitudinal relaxation depending on the fibrillation stage of Aβ, because molecular tumbling rate of the Gd complexes becomes slower (Fig. 1A).¹⁷ As a result, the probes permit the detection of Aβ by longitudinal relaxation time (T₁)-weighted imaging. This mechanism could also be utilized to estimate the inhibitory activity of the probes by T₁-based analysis (Fig. 1B). The curcumin and compound B were directly conjugated with the macrocyclic DO3A ligand through the propylamine linker to obtain Gd-DO3A-Cur and Gd-DO3A-Comp.B, respectively (Fig. 1C).Open in a separate windowFig. 1(A) A probe concept that produces T₁ in a dependent manner of Aβ fibrillation process. (B) Inhibitor-based probes that cause moderate T₁ decreases due to inhibitory activity of fibrillation. (C) The chemical structures of the synthesized Gd probes for Aβ detection and inhibition.Gd-DO3A-Cur and Gd-DO3A-Comp.B were synthesized according to Scheme 1 (detail in Scheme S1, ESI^†). The compound 5a and 5b, which have asymmetric curcumin derivatives containing carboxylic acid group, were synthesized by three step reactions. Amide bond formation with DO3A(tBu)₃-propylamine ligand¹⁸ by condensation reaction afforded compound 7a and 7b. The tert-butyl groups were deprotected by trifluoroacetic acid producing compound 8a and 8b. The complexation was performed with GdCl₃·6H₂O by adjusting the reaction pH to 7, giving 43 and 41% yields of Gd-DO3A-Cur and Gd-DO3A-Comp.B, respectively. The T₁ relaxivities (r₁) of the curcumin-based Gd probes were estimated by T₁ measurement using a 1 tesla NMR relaxometry (Fig. S1, ESI^†). For the comparison, we synthesized Gd-DO3A-Chal which is a reported probe for Aβ.¹⁹ The r₁ of Gd-DO3A-Comp.B, Gd-DO3A-Cur, and Gd-DO3A-Chal were 7.1, 6.1 and 5.3 mM⁻¹ s⁻¹, respectively. These r₁ values are higher than that of clinically approved Gd-DOTA (3.9 mM⁻¹ s⁻¹).²⁰ The molecular weight of Gd-DO3A-Comp.B and Gd-DO3A-Cur is almost two times larger than that of Gd-DOTA. Because the r₁ increases approximately linearly with molecular weight in low magnetic field,¹⁷ the high r₁ values of Gd-DO3A-Comp.B and Gd-DO3A-Cur might be mainly attributed to their high rotational correlation time, rather than the high number of coordinated water molecules. The r₁ of Gd-DO3A-Chal was comparable to the value reported previously.¹⁹Open in a separate windowScheme 1Synthetic scheme of Gd-DO3A-Cur and Gd-DO3A-Comp.B. (a) B(OH)₃, morpholine, DMF, 100 °C, 10 min. (b) 3a/3b, B(OH)₃, morpholine, DMF, 100 °C, 10 min. (c) TFA, DCM. (d) DO3A(tBu)₃-propylamine ligand, PyBOP, HOBt, Et₃N, DMF. (e) 7a/7b, TFA, DCM. (f) GdCl₃·6H₂O, NaOH, H₂O.We evaluated the inhibitory effect of three probes toward Aβ aggregation by Congo red assay.²¹ After 24 h incubation of 20 μM Aβ with 10 μM probe, Gd-DO3A-Comp.B showed the lowest fluorescence intensity, indicating the strongest inhibitory activity followed by Gd-DO3A-Cur (Fig. 2A). As the comparison, the reported MRI agents, Gd-DO3A-Chal showed slight inhibitory activity. The inhibitory effect was further evaluated by transmission electron microscopy (TEM) with negative staining (Fig. 2B). In the absence of the probes, Aβ formed huge and massive fibril similar to the typical morphology of Aβ fibril.²² The TEM images of Aβ with Gd-DO3A-Comp.B showed the presence of white spheres below 10 nm, demonstrating that Gd-DO3A-Comp.B strongly inhibits Aβ aggregation. In fact, the fibril growth stopped at a stage of oligomer formation. Lower inhibitory activity of Gd-DO3A-Cur was also found to provide a shortened worm-like fibril, which is the typical morphology of Aβ exposed to curcumin.²³ In contrast, the small amount of white spheres and partial fibril disruption were found in the image of Aβ with Gd-DO3A-Chal. In comparison with a reported Gd-DTPA-curcumin possessing inhibitory activity starting at 50 μM, Gd-DO3A-Comp.B possessed stronger inhibition of Aβ aggregation at 10 μM.²⁴ The MTT assay using Neuro 2a cells showed that IC₅₀ of Gd-DO3A-Cur and Gd-DO3A-Comp.B. were more than 500 μM, indicating that these compounds did not possess significant cytotoxicity (Fig. S2, ESI^†).Open in a separate windowFig. 2Inhibitory effect of the Gd probes toward Aβ aggregation measured by Congo red assay (A) and negative staining TEM images (B). The Gd probes were co-incubated with monomeric Aβ for 24 h in PBS at pH 7.4. [Gd] = 10 μM, [Aβ] = 20 μM. Scale bars = 100 nm.To detect fibrillation process by NMR relaxometry, we measured T₁ of the probe mixture with Aβ which were pre-incubated for 1, 3, 6, 12, and 24 h to make it form the fibrils of different growth stages (Fig. 3A and B). The T₁ of Gd-DO3A-Comp.B solution decreased with pre-incubation time of Aβ, demonstrating that the Gd-DO3A-Comp.B can detect Aβ fibril depending on the growth stage (Fig. 3B). Lower T₁ involved with Aβ growth could be caused by the reduction in tumbling rate of the Gd complex.²⁵ We also co-incubated the probes with the Aβ monomer and monitored T₁ changes over the incubation time (Fig. 3A, B and S3, ESI^†). Interestingly, the Gd-DO3A-Comp.B did not cause significant T₁ decreases even after 24 hours co-incubation with Aβ monomers, demonstrating that Gd-DO3A-Comp.B has a strong inhibitory effect on fibril formation and the inhibition can be monitored by T₁ measurement (Fig. 3B). The inhibitory effect was consistent with the results of Congo red assay and TEM (Fig. 2). On the other hand, the time-dependent increases of T₁ were observed in Gd-DO3A-Chal and Gd-DO3A-Cur. This might be because these two probes were buried in the hydrophobic pocket as Aβ fibril grew up and fewer water molecules permitted access to the Gd ions. It is also possible that these probes have lower binding affinity, especially for matured fibril, and require higher concentrations to produce significant T₁ changes.²⁶ These probe did not produce the significant ΔT₁ between monomer and fibril samples (Fig. 3B and S3, ESI^†), although they showed little inhibition in Congo red assay and TEM (Fig. 2).Open in a separate windowFig. 3(A) Experimental design of T₁-based detection of Aβ fibrillation and inhibition by using the Gd probes. (B) T₁ changes of the Gd probe solutions with pre-incubated fibrils and monomers in PBS at pH 7.4 (mean ± SEM, n = 3). [Gd] = 10 μM, [Aβ] = 20 μM.The feasibility of the Gd probes was further evaluated by in vitro MRI measurement using a 1 tesla scanner. The T₁-weighted images showed that Gd-DO3A-Comp.B produced slight T₁ signal increases with Aβ monomers for 2 and 24 h (Fig. 4A and B). More significant signal increases were observed in the Gd-DO3A-Comp.B with Aβ fibril pre-incubated for 24 h (Fig. 4C). In contrast, Gd-DO3A-Chal and Gd-DO3A-Cur did not show significant signal changes in the presence of Aβ monomers or fibrils (Fig. 4A–C). These results were mostly consistent with the T₁ profile measured by NMR (Fig. 3). Compared to the previously reported Gd-DO3A-Chal that required 100 μM of the probe concentration to detect the equimolar Aβ,¹⁹ Gd-DO3A-Comp.B could detect five-times lower concentration of Aβ (20 μM) with ten-times lower probe concentration (10 μM). Therefore, Gd-DO3A-Comp.B could be promising to further develop highly sensitive diagnostic MRI contrast agents of AD.Open in a separate windowFig. 4 T ₁-weighted images of the Gd probe solutions in the presence of monomeric Aβ at 2 h incubation (A), monomeric Aβ at 24 h incubation (B), and Aβ fibrils pre-incubated for 24 h (C). Incubation was conducted in PBS at pH 7.4.In conclusion, we synthesized the curcumin-based Gd probes which enabled the detection and inhibition of Aβ fibril formation. Gd-DO3A-Comp.B allowed for the highly sensitive detection of Aβ fibril by the T₁ measurement. Moreover, the inhibitory activity could be estimated by T₁ measurement, because Gd-DO3A-Comp.B decreased T₁ depending on the growth stage of Aβ fibril formation. Such unique modality would be useful not only for the diagnostics but also for the direct evaluation of the therapeutic efficacy in vivo. For the future application, it would be important to combine with BBB penetration methods targeting the brain such as transient opening of the BBB using focused ultrasound or mannitol injection.^27,28