A Simple and Robust Exponential Amplification Reaction (EXPAR)-Based Hairpin Template (exp-Hairpin) for Highly Specific, Sensitive, and Universal MicroRNA Detection.

Specific and sensitive detection of microRNAs continues to encounter significant challenges, especially in the development of rapid and efficient isothermal amplification strategies for point-of-care settings. The exponential amplification reaction (EXPAR) has garnered significant attention owing to its simplicity and rapid amplification of signals within a short period. However, a substantial loss of amplification efficiency, difficulty in distinguishing closely related homologous sequences, and adapting the designed templates to other targets seriously hamper the practical application of the EXPAR. In this work, a hairpin template tailored for the EXPAR system (exp-Hairpin) was constructed by adding identical trigger sequences and enzyme cleavage sites on two arms of the hairpin, achieving theoretically more than 2n amplification efficiency and minimal background amplification of EXPAR. Modulating the stability of the exp-Hairpin template by increasing the stem length, the specificity of detecting target miRNA in highly homologous sequences could be significantly improved. Using miRNA let-7a as a target model, the exp-Hairpin with 8 bp stem length for EXPAR amplification curves could effectively distinguish target let-7a and nontarget let-7b/7c/7f/7g/7i homologous sequences. This strategy enabled the sensitive and accurate analysis of let-7a in diluted human serum with satisfactory recoveries. By simply replacing the loop recognition sequence of exp-Hairpin, the specific detection of miR-200b was also achieved, demonstrating the universality of this strategy. The exp-Hairpin EXPAR accelerates simple and rapid molecular diagnostic applications for short nucleic acids.

Importin α phosphorylation promotes TPX2 activation by GM130 to control astral microtubules and spindle orientation.

Spindle orientation is important in multiple developmental processes as it determines cell fate and function. The orientation of the spindle depends on the assembly of a proper astral microtubule network. Here, we report that the spindle assembly factor TPX2 regulates astral microtubules. TPX2 in the spindle pole area is activated by GM130 (GOLGA2) on Golgi membranes to promote astral microtubule growth. GM130 relieves TPX2 inhibition by competing for importin α1 (KPNA2) binding. Mitotic phosphorylation of importin α at serine 62 (S62) by CDK1 switches its substrate preference from TPX2 to GM130, thereby enabling competition-based activation. Importin α S62A mutation impedes local TPX2 activation and compromises astral microtubule formation, ultimately resulting in misoriented spindles. Blocking the GM130-importin α-TPX2 pathway impairs astral microtubule growth. Our results reveal a novel role for TPX2 in the organization of astral microtubules. Furthermore, we show that the substrate preference of the important mitotic modulator importin α is regulated by CDK1-mediated phosphorylation.

Low-Triggering-Potential Electrochemiluminescence from a Luminol Analogue Functionalized Semiconducting Polymer Dots for Imaging Detection of Blood Glucose.

In recent years, semiconducting polymer dots (Pdots) as environmentally friendly and high-brightness electrochemiluminescence (ECL) nanoemitters have attracted intense attention in ECL biosensing and imaging. However, most of the available Pdots have a high ECL excitation potential in the aqueous phase (>1.0 V vs Ag/AgCl), which causes poor selectivity in actual sample detection. Therefore, it is particularly important to construct a simple and universal strategy to lower the trigger potential of Pdots. This work has realized the ECL emission of Pdots at low-trigger-potential based on the electrochemiluminescence resonance energy transfer (ERET) strategy. By covalently coupling the Pdots with a luminol analogue, N-(4-aminobutyl)-N-ethylisoluminol (ABEI), the ABEI-Pdots showed an anodic ECL emission with a low onset potential of +0.34 V and a peak potential at +0.45 V (vs Ag/AgCl), which was the lowest trigger potential reported so far. We further explored this low-triggering-potential ECL for imaging detection of glucose in buffer and serum. By imaging the ABEI-Pdots-modified screen-printed electrodes (SPCE) at +0.45 V for 16 s, the ECL imaging method could quantify the glucose concentration in buffer from 10 to 200 µM with detection limits of 3.3 µM, while exhibiting excellent selectivity. When applied to real serum, the results of our method were highly consistent with a commercial blood glucose meter, with the relative errors ranging from 3.2 to 13%. This work provided a universal strategy for constructing low potential Pdots and demonstrated its application potential in complex biological sample analysis.

Synthesis and screening of 3-MA derivatives for autophagy inhibitors.

Autophagy is a conserved degradation process, which plays important pathophysiological roles. The lack of effective inhibitors of autophagy has been an obstacle in both basic research and understanding the physiological role of autophagy in disease manifestation. The most widely used inhibitor, 3-methyladenine (3-MA), is poorly soluble at room temperature and is effective only at high concentrations. In this study, we synthesized a library of small compounds by chemically modifying 3-MA and screened this library for autophagy inhibitors. Three 3-MA derivatives generated through this approach showed improved solubility and effectiveness in inhibiting autophagy. We demonstrated that chemical modification of an existing autophagy inhibitor is an effective method to generate improved autophagy inhibitors.