Optical lock-in detection of FRET using synthetic and genetically encoded optical switches

The Förster resonance energy transfer (FRET) technique is widely used for studying protein interactions within live cells. The effectiveness and sensitivity of determining FRET, however, can be reduced by photobleaching, cross talk, autofluorescence, and unlabeled, endogenous proteins. We present a FRET imaging method using an optical switch probe, Nitrobenzospiropyran (NitroBIPS), which substantially improves the sensitivity of detection to <1% FRET efficiency. Through orthogonal optical control of the colorful merocyanine and colorless spiro states of the NitroBIPS acceptor, donor fluorescence can be measured both in the absence and presence of FRET in the same FRET pair in the same cell. A SNAP-tag approach is used to generate a green fluorescent protein-alkylguaninetransferase fusion protein (GFP-AGT) that is labeled with benzylguanine-NitroBIPS. In vivo imaging studies on this green fluorescent protein-alkylguaninetransferase (GFP-AGT) (NitroBIPS) complex, employing optical lock-in detection of FRET, allow unambiguous resolution of FRET efficiencies below 1%, equivalent to a few percent of donor-tagged proteins in complexes with acceptor-tagged proteins.     

金门岛北部海域浮游植物的季节变动及与环境的关联

采用2013—2014年四季度月在金门岛北部海域获取的浮游植物及环境因子监测数据, 分析该区浮游植物的群落结构和季节变化及其与温度、盐度、悬浮物、营养盐、叶绿素等的关系, 初步探讨涉海工程建设对浮游植物群落的潜在影响。结果显示, 鉴定出的浮游植物隶属3门43属82种(不含未定种), 群落构成以硅藻为主, 其次是甲藻, 蓝藻仅1种。物种组成的季节差异较大, 3月物种贫乏, 1月次之, 7月和11月最丰富。四季丰度平均为47.09×103 cells/L, 1月丰度最高, 7月次之, 11月最低, 3月高于11月少许。四季优势种均为硅藻, 13个优势种分别为柔弱几内亚藻(Guinardia delicatula)、短角弯角藻(Ecampia zoodicaus)、骨条藻(Skeletonema spp.)、具槽帕拉藻(Paralia sulcata)、微小海链藻(Thalassiosira exigua)、标志星杆藻(Asterionella notula)、旋链角毛藻(Chaetoceros curvisetus)、新月菱形藻(Nitzchia closterium)、派格棍形藻(Bacillaria paxillifera)、异常角毛藻(Chaetoceros abmormis)、小细柱藻(Leptocylindrus minutum)、宽角曲舟藻(Pleurosigma angulatum)和美丽曲舟藻(Pleurosigma formosum)。不同季节优势种有一定程度交错, 仅在单季占优的有6种, 有2/3在3个以上季节出现, 具槽帕拉藻、骨条藻为四季优势种。浮游植物物种多样性和均匀度总体较好, 群落结构稳定。与毗邻海区相比, 本区物种丰富度偏低, 丰度高于毗邻海区, 种类组成相似, 优势种却有较大差别。Pearson相关分析表明, 溶解无机氮及活性磷酸盐仅在1月与丰度存在极显著的正相关, 是促使丰度为四季最高的原因。涉海工程施工产生的悬浮物和冲击波是影响浮游植物群落的主要因素, 大量海洋工程建设案例表明, 施工期造成的浮游植物丰度下降趋势和优势种更替混乱在工程结束后能得以恢复。     

选择性支气管动脉插管转铁蛋白受体单克隆抗体灌注治疗中晚期肺癌(附68例临床报告)

本文对68例中晚期肺癌进行选择性支气管动脉插管采用转铁蛋白受体单克隆抗体与表阿霉素、顺伯等药物制成偶联物灌注。结果显示,肿瘤明显消退24例(35.2％),部分消退36例(52.5％),无变化8例(12.3％),总有效率为87.7％。通过对支气管动脉插管,导向灌注疗法结果显示,本疗法优于周围静脉供药,且无严重并发症,副作用轻,不失为一种可供选择的治疗方法。     

ART患者早期流产组织染色体异常及相关因素分析

目的:分析ART患者早期流产组织染色体异常及其相关影响因素。方法:回顾性分析2013-2017年ART患者行早期流产组织染色体检查的409例样本,分析胚胎染色体非整倍性发生及其与女方年龄、不孕年限、不孕因素、促排卵指标之间的关系。结果:ART流产患者中,流产组织染色体非整倍性发生率为57.46%,发生频次以16三体占比最高(23.95%),其次是22三体(13.45%)及Turner(9.24%)。流产组织染色体非整倍性患者平均年龄高于染色体整倍性患者(P0.001)。16三体组患者年龄低于22三体(P0.01)及Turner组(P0.05)。16三体组患者平均Gn使用量低于22三体组(P0.05)。16三体组患者移植15天血HCG值低于22三体(P0.05)及Turner组(P0.01)。结论:ART患者流产组织染色体非整倍性与女方年龄正相关,但16三体及Turner的发生与女方年龄相关性不大,且16三体更容易引发早期流产。     

Isolation and characterization of a human novel RAB (RAB39B) gene

Rab proteins are small-molecular-weight GTPases that control vesicular trafficking in eukaryotic cells. During the large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a cDNA clone encoding a novel Rab protein, which showed 74.2% identity with previously isolated Rab39A at the amino acid level. RAB39B was expressed in a variety of human tissues and located in human chromosome Xq28. It consisted of two exons spanning 3764 bp of human genomic DNA.     

重组人IL6／2融合蛋白对6.5Gy照射小鼠造血功能恢复的影响

观察了ｈＦＰＩＬ６／２对６．５Ｇｙγ线照射ＮＩＨ小鼠第１０天造血功能恢复的影响。结果表明：照射小鼠连续４ｄ给予ｈＦＰＩＬ６／２２５０μｇ·ｋｇ－１·ｄ－１,其脾重、ＣＦＵ－８、骨髓有核细胞数及ＣＭ－ＣＦＵ分别比对照组增加５９．０％、２７８．５％、５７．９％和１３８．２％,统计学处理均有显著差异;对此四项指标的改善也明显优于２５μｇ组。另外,２５０μｇ剂量组小鼠外周血象３０ｄ的动态观察结果表明,ｈＦＰＩＬ６／２不但能明显提高红细胞和血红蛋白的最低值,而且能使血小板的恢复提前。提示ｈＦＰＩＬ６／２在促进血小板生成和促进红系造血方面可能具有良好的应用前景。     

Secretion of annexin A3 from ovarian cancer cells and its association with platinum resistance in ovarian cancer patients

Early detection of resistance to platinum-based therapy is critical for improving the treatment of ovarian cancers. We have previously found that increased expression of annexin A3 is a mechanism for platinum resistance in ovarian cancer cells. Here we demonstrate that annexin A3 can be detected in the culture medium of ovarian cancer cells, particularly these cells that express high levels of annexin A3. Levels of annexin A3 were then determined in sera from ovarian cancer patients using an enzyme-linked immunosorbent assay. Compared with those from normal donors, sera from ovarian cancer patients contain significantly higher levels of annexin A3. Furthermore, serum levels of annexin A3 were significantly higher in platinum-resistant patients than in platinum-sensitive patients. To gain insight into the mechanism of secretion, the ovarian cancer cell lines were examined using both transmission electron microscopy and immunoelectron microscopy. Compared with parent cells, there are significantly more vesicles in the cytoplasm of ovarian cancer cells that express high levels of annexin A3, and at least some vesicles are annexin A3-positive. Moreover, some vesicles appear to be fused with the cell membrane, suggesting that annexin A3 secretion may be associated with exocytosis and the release of exosomes. This is supported by our observation that ovarian cancer cells expressing higher levels of annexin A3 released increased numbers of exosomes. Furthermore, annexin A3 can be detected in exosomes released from cisplatin-resistant cells (SKOV3/Cis) by immunoblotting and immunoelectron microscopy.     

Autophagic Impairment Contributes to Systemic Inflammation-Induced Dopaminergic Neuron Loss in the Midbrain

Background

Neuroinflammation plays an important role in the pathogenesis of Parkinson’s disease (PD), inducing and accelerating dopaminergic (DA) neuron loss. Autophagy, a critical mechanism for clearing misfolded or aggregated proteins such as α-synuclein (α-SYN), may affect DA neuron survival in the midbrain. However, whether autophagy contributes to neuroinflammation-induced toxicity in DA neurons remains unknown.

Results

Intraperitoneal injection of lipopolysaccharide (LPS, 5 mg/kg) into young (3-month-old) and aged (16-month-old) male C57BL/6J mice was observed to cause persistent neuroinflammation that was associated with a delayed and progressive loss of DA neurons and accumulation of α-SYN in the midbrain. The autophagic substrate-p62 (SQSTM1) persistently increased, whereas LC3-II and HDAC6 exhibited early increases followed by a decline. In vitro studies further demonstrated that TNF-α induced cell death in PC12 cells. Moreover, a sublethal dose of TNF-α (50 ng/ml) increased the expression of LC3-II, p62, and α-SYN, implying that TNF-α triggered autophagic impairment in cells.

Conclusion

Neuroinflammation may cause autophagic impairment, which could in turn result in DA neuron degeneration in midbrain.     

Structural Insight into Layer Gliding and Lattice Distortion in Layered Manganese Oxide Electrodes for Potassium‐Ion Batteries

Potassium‐ion batteries (PIBs) are an emerging, affordable, and environmentally friendly alternative to lithium‐ion batteries, with their further development driven by the need for suitably performing electrode materials capable of reversibly accommodating the relatively large K⁺. Layer‐structured manganese oxides are attractive as electrodes for PIBs, but suffer from structural instability and sluggish kinetics of K⁺ insertion/extraction, leading to poor rate capability. Herein, cobalt is successfully introduced at the manganese site in the K_xMnO₂ layered oxide electrode material and it is shown that with only 5% Co, the reversible capacity increases by 30% at 22 mA g^‐1 and by 92% at 440 mA g^‐1. In operando synchrotron X‐ray diffraction reveals that Co suppresses Jahn–Teller distortion, leading to more isotropic migration pathways for K⁺ in the interlayer, thus enhancing the ionic diffusion and consequently, rate capability. The detailed analysis reveals that additional phase transitions and larger volume change occur in the Co‐doped material as a result of layer gliding, with these associated with faster capacity decay, despite the overall capacity remaining higher than the pristine material, even after 500 cycles. These results assert the importance of understanding the detailed structural evolution that underpins performance that will inform the strategic design of electrode materials for high‐performance PIBs.