Real-Time analysis of exosome secretion of single cells with single molecule imaging

The exosome-mediated response can promote or restrain the diseases by regulating the intracellular pathways, making the exosome become an effective marker for diagnosis and therapeutic control at the single-cell level. However, real-time analysis is hard to be achieved with traditional approaches because the exosomes usually need to be enriched by ultracentrifugation for a measurable signal-to-noise ratio. Recently developed label-free single-molecule imaging approaches may become an real-time quantitative tool for the analysis of single exosomes and related secretion behaviors of single living cells owing to their extreme sensitivity.     

Preclinical Evaluation of 99mTc-ZHER2:41071, a Second-Generation Affibody-Based HER2-Visualizing Imaging Probe with a Low Renal Uptake

Radionuclide imaging of HER2 expression in tumours may enable stratification of patients with breast, ovarian, and gastroesophageal cancers for HER2-targeting therapies. A first-generation HER2-binding affibody molecule [^99mTc]Tc-ZHER2:V2 demonstrated favorable imaging properties in preclinical studies. Thereafter, the affibody scaffold has been extensively modified, which increased its melting point, improved storage stability, and increased hydrophilicity of the surface. In this study, a second-generation affibody molecule (designated ZHER2:41071) with a new improved scaffold has been prepared and characterized. HER2-binding, biodistribution, and tumour-targeting properties of [^99mTc]Tc-labelled ZHER2:41071 were investigated. These properties were compared with properties of the first-generation affibody molecules, [^99mTc]Tc-ZHER2:V2 and [^99mTc]Tc-ZHER2:2395. [^99mTc]Tc-ZHER2:41071 bound specifically to HER2 expressing cells with an affinity of 58 ± 2 pM. The renal uptake for [^99mTc]Tc-ZHER2:41071 and [^99mTc]Tc-ZHER2:V2 was 25–30 fold lower when compared with [^99mTc]Tc-ZHER2:2395. The uptake in tumour and kidney for [^99mTc]Tc-ZHER2:41071 and [^99mTc]Tc-ZHER2:V2 in SKOV-3 xenografts was similar. In conclusion, an extensive re-engineering of the scaffold did not compromise imaging properties of the affibody molecule labelled with ^99mTc using a GGGC chelator. The new probe, [^99mTc]Tc-ZHER2:41071 provided the best tumour-to-blood ratio compared to HER2-imaging probes for single photon emission computed tomography (SPECT) described in the literature so far. [^99mTc]Tc-ZHER2:41071 is a promising candidate for further clinical translation studies.     

Alpha Synuclein only Forms Fibrils In Vitro when Larger than its Critical Size of 70 Monomers

The aggregation of α-synuclein into small soluble aggregates and then fibrils is important in the development and spreading of aggregates through the brain in Parkinson's disease. Fibrillar aggregates can grow by monomer addition and then break into fragments that could spread into neighboring cells. The rate constants for fibril elongation and fragmentation have been measured but it is not known how large an aggregate needs to be before fibril formation is thermodynamically favorable. This critical size is an important parameter controlling at what stage in an aggregation reaction fibrils can form and replicate. We determined this value to be approximately 70 monomers using super-resolution and atomic force microscopy imaging of individual α-synuclein aggregates formed in solution over long time periods. This represents the minimum size for a stable α-synuclein fibril and we hypothesis the formation of aggregates of this size in a cell represents a tipping point at which rapid replication occurs.     

盖子环替换及定点突变提高菜豆环氧化物水解酶对邻甲基苯基缩水甘油醚的催化性能

菜豆环氧化物水解酶1和2（PvEH1、PvEH2）能够动力学拆分外消旋邻甲基苯基缩水甘油醚（rac-oMGE），从而保留(R)-oMGE。基于对PvEH1和PvEH2结构的同源模拟和分析，发现二者分子中的盖子环差异较大，故本文选择盖子环作为研究目标。经融合聚合酶链式反应（FPCR），获得了PvEH2的盖子环区域被PvEH1对应区域替换的杂合酶Pv2Pv1。用全细胞酶E. coli/pv2pv1催化rac-oMGE，当(S)-oMGE刚好水解完全时，产物(S)-3-邻甲苯基-1,2-丙二醇（(S)-oTPD）的ee_p由PvEH2的58.3%提高至75.5%。为进一步提高酶的性质，在Pv2Pv1中选取11个氨基酸位点进行丙氨酸(A)突变，获得最优突变子E. coli/pv2pv1^K176A，活性为E. coli/pv2pv1（4.2U/g）的2.1倍，且当S构型的底物刚好完全水解时，(S)-oTPD的ee_p进一步提高为80.3%。分子对接分析发现，盖子环替换和K176位点突变为A，均使(R)-oMGE环氧环中的C_α更易受到酶中D101位点的攻击。利用E. coli/pv2pv1^K176A催化150mmol/L rac-oMGE水解制备(R)-oMGE（ee_s＞99%）和(S)-oTPD（ee_p=80.4%），二者的产率Y_S和Y_P分别为32.7%和60.1%，时空产率STY_S和STY_P为1.6g/(L·h)和3.3g/(L·h)。本实验为改善EH的催化性质提供了一种有效策略。     

Deep blue/ultraviolet microcavity OLEDs based on solution-processed PVK:CBP blends

There is an increasing need to develop stable, high-intensity, efficient OLEDs in the deep blue and UV. Applications include blue pixels for displays and tunable narrow solid-state UV sources for sensing, diagnostics, and development of a wide band spectrometer-on-a-chip. With the aim of developing such OLEDs we demonstrate an array of deep blue to near UV tunable microcavity (μc) OLEDs (λ ∼373–469 nm) using, in a unique approach, a mixed emitting layer (EML) of poly(N-vinyl carbazole) (PVK) and 4,4′-bis(9-carbazolyl)-biphenyl (CBP), whose ITO-based devices show a broad electroluminescence (EL) in the wavelength range of interest. This 373–469 nm band expands the 493–640 nm range previously attained with μcOLEDs into the desired deep blue-to-near UV range. Moreover, the current work highlights interesting characteristics of the complexity of mixed EML emission in combinatorial 2-d μcOLED arrays of the structure 40 nm Ag/x  nm MoO_x/∼30 nm PVK:CBP (3:1 weight ratio)/y  nm 4,7-diphenyl-1,10-phenanthroline (BPhen)/1 nm LiF/100 nm Al, where x = 5, 10, 15, and 20 nm and y = 10, 15, 20, and 30 nm. In the short wavelength μc devices, only CBP emission was observed, while in the long wavelength μc devices the emission from both PVK and CBP was evident. To understand this behavior simulations based on the scattering matrix method, were performed. The source profile of the EML was extracted from the measured EL of ITO-based devices. The calculated μc spectra indeed indicated that in the thinner, short wavelength devices the emission is primarily from CBP; in the thicker devices both CBP and PVK contribute to the EL. This situation is due to the effect of the optical cavity length on the relative contributions of PVK and CBP EL through a change in the wavelength-dependent emission rate, which was not suggested previously. Structural analysis of the EML and the preceding MoO_x layer complemented the data analysis.     

Allele-Specific Inhibition of Histone Demethylases

Histone demethylases play a critical role in mammalian gene expression by removing methyl groups from lysine residues in degree- and site-specific manner. To specifically interrogate members and isoforms of this class of enzymes, we have developed demethylase variants with an expanded active site. The mutant enzymes are capable of performing lysine demethylation with wild-type proficiency, but are sensitive to inhibition by cofactor-competitive molecules embellished with a complementary steric “bump”. The selected inhibitors show more than 20-fold selectivity over the wild-type demethylase, thus overcoming issues typical to pharmacological and genetic approaches. The mutant–inhibitor pairs are shown to act on a physiologically relevant full-length substrate. By engineering a conserved amino acid to achieve member-specific perturbation, this study provides a general approach for studying histone demethylases in diverse cellular processes.     

“动态超分子”假说预测烷烃吸收CO2相平衡

采用“动态超分子假说”对烷烃吸收CO₂相平衡进行了研究,并结合最小二乘法和试算对烷烃数据进行拟合,得到相关参数,拟合值和文献实验值的线性相关度均大于0.95,符合良好。研究表明,烷烃分子之间形成的同种“动态超分子”对吸收过程影响较大,远远大于CO₂同种超分子和溶剂与CO₂之间形成的异种超分子,而且其随着烷烃碳链增加,“动态超分子”形成概率和稳定性均降低,吸收能力逐渐增强。文中还研究了模型参数对拟合结果的影响,及其最佳取值范围。