ZW10: an emerging orchestrator of organelle dynamics during the cell division cycle.

Zeste white 10 (ZW10) was first identified as a centromere/kinetochore protein encoded by the ZW10 gene in Drosophila. ZW10 guides the spindle assembly checkpoint signaling during mitotic chromosome segregation in metazoans. Recent studies have shown that ZW10 is also involved in membranous organelle interactions during interphase and plays a vital role in membrane transport between the endoplasmic reticulum and Golgi apparatus. Despite these findings, the precise molecular mechanisms by which ZW10 regulates interactions between membranous organelles in interphase and the assembly of membraneless organelle kinetochore in mitosis remain elusive. Here, we highlight how ZW10 forms context-dependent protein complexes during the cell cycle. These complexes are essential for mediating membrane trafficking in interphase and ensuring the accurate segregation of chromosomes in mitosis.

MicroRNA-10 Family Promotes Renal Fibrosis through the VASH-1/Smad3 Pathway.

Renal fibrosis (RF) stands as a pivotal pathological process in the advanced stages of chronic kidney disease (CKD), and impeding its progression is paramount for delaying the advancement of CKD. The miR-10 family, inclusive of miR-10a and miR-10b, has been implicated in the development of various fibrotic diseases. Nevertheless, the precise role of miR-10 in the development of RF remains enigmatic. In this study, we utilized both an in vivo model involving unilateral ureteral obstruction (UUO) in mice and an in vitro model employing TGF-ß1 stimulation in HK-2 cells to unravel the mechanism underlying the involvement of miR-10a/b in RF. The findings revealed heightened expression of miR-10a and miR-10b in the kidneys of UUO mice, accompanied by a substantial increase in p-Smad3 and renal fibrosis-related proteins. Conversely, the deletion of these two genes led to a notable reduction in p-Smad3 levels and the alleviation of RF in mouse kidneys. In the in vitro model of TGF-ß1-stimulated HK-2 cells, the co-overexpression of miR-10a and miR-10b fostered the phosphorylation of Smad3 and RF, while the inhibition of miR-10a and miR-10b resulted in a decrease in p-Smad3 levels and RF. Further research revealed that miR-10a and miR-10b, through binding to the 3'UTR region of Vasohibin-1 (VASH-1), suppressed the expression of VASH-1, thereby promoting the elevation of p-Smad3 and exacerbating the progression of RF. The miR-10 family may play a pivotal role in RF.

Label-free active single-cell encapsulation enabled by microvalve-based on-demand droplet generation and real-time image processing.

Droplet microfluidics-based single-cell encapsulation is a critical technology that enables large-scale parallel single-cell analysis by capturing and processing thousands of individual cells. As the efficiency of passive single-cell encapsulation is limited by Poisson distribution, active single-cell encapsulation has been developed to theoretically ensure that each droplet contains one cell. However, existing active single-cell encapsulation technologies still face issues related to fluorescence labeling and low throughput. Here, we present an active single-cell encapsulation technique by using microvalve-based drop-on-demand technology and real-time image processing to encapsulate single cells with high throughput in a label-free manner. Our experiments demonstrated that the single-cell encapsulation system can encapsulate individual polystyrene beads with 96.3 % efficiency and HeLa cells with 94.9 % efficiency. The flow speed of cells in this system can reach 150 mm/s, resulting in a corresponding theoretical encapsulation throughput of 150 Hz. This technology has significant potential in various biomedical applications, including single-cell omics, secretion detection, and drug screening.

Aurora-A condensation mediated by BuGZ aids its mitotic centrosome functions.

Centrosomes composed of centrioles and the pericentriolar material (PCM), serve as the platform for microtubule polymerization during mitosis. Despite some centriole and PCM proteins have been reported to utilize liquid-liquid phase separation (LLPS) to perform their mitotic functions, whether and how centrosomal kinases exert the coacervation in mitosis is still unknown. Here we reveal that Aurora-A, one key centrosomal kinase in regulating centrosome formation and functions, undergoes phase separation in vitro or in centrosomes from prophase, mediated by the conserved positive-charged residues inside its intrinsic disordered region (IDR) and the intramolecular interaction between its N- and C-terminus. Aurora-A condensation affects centrosome maturation, separation, initial spindle formation from the spindle pole and its kinase activity. Moreover, BuGZ interacts with Aurora-A to enhance its LLPS and centrosome functions. Thus, we propose that Aurora-A collaborates with BuGZ to exhibit the property of LLPS in centrosomes to control its centrosome-dependent functions from prophase.

Amygdala-Targeted Relief of Neuropathic Pain: Efficacy of Repetitive Transcranial Magnetic Stimulation in NLRP3 Pathway Suppression.

This study investigates the effectiveness of repetitive transcranial magnetic stimulation (rTMS) as a nonpharmacological approach to treating neuropathic pain (NP), a major challenge in clinical research. Conducted on male Sprague-Dawley rats with NP induced through chronic constriction injury of the sciatic nerve, the research assessed pain behaviors and the impact of rTMS on molecular interactions within the amygdala. Through a comprehensive analysis involving Mechanical Withdrawal Threshold (MWT), Thermal Withdrawal Latency (TWL), RNA transcriptome sequencing, RT-qPCR, Western blotting, immunofluorescence staining, and Co-Immunoprecipitation (Co-IP), the study focused on the expression and interaction of integrin αvß3 and its receptor P2X7R. Findings reveal that rTMS significantly influences the expression of integrin αvß3 in NP models, suggesting an inhibition of the NP-associated NLRP3 inflammatory pathway through the disruption of integrin αvß3-P2X7R interactions. These outcomes highlight the potential of rTMS in alleviating NP by targeting molecular interactions within the amygdala, offering a promising therapeutic avenue for managing NP.

Organization of microtubule plus-end dynamics by phase separation in mitosis.

In eukaryotes, microtubule polymers are essential for cellular plasticity and fate decisions. End-binding (EB) proteins serve as scaffolds for orchestrating microtubule polymer dynamics and are essential for cellular dynamics and chromosome segregation in mitosis. Here, we show that EB1 forms molecular condensates with TIP150 and MCAK through liquid-liquid phase separation to compartmentalize the kinetochore-microtubule plus-end machinery, ensuring accurate kinetochore-microtubule interactions during chromosome segregation in mitosis. Perturbation of EB1-TIP150 polymer formation by a competing peptide prevents phase separation of the EB1-mediated complex and chromosome alignment at the metaphase equator in both cultured cells and Drosophila embryos. Lys220 of EB1 is dynamically acetylated by p300/CBP-associated factor in early mitosis, and persistent acetylation at Lys220 attenuates the phase separation of the EB1-mediated complex, dissolves droplets in vitro, and harnesses accurate chromosome segregation. Our data suggest a novel framework for understanding the organization and regulation of eukaryotic spindle for accurate chromosome segregation in mitosis.

CSPP1 stabilizes microtubules by capping both plus and minus ends.

Although the dynamic instability of microtubules (MTs) is fundamental to many cellular functions, quiescent MTs with unattached free distal ends are commonly present and play important roles in various events to power cellular dynamics. However, how these free MT tips are stabilized remains poorly understood. Here, we report that centrosome and spindle pole protein 1 (CSPP1) caps and stabilizes both plus and minus ends of static MTs. Real-time imaging of laser-ablated MTs in live cells showed deposition of CSPP1 at the newly generated MT ends, whose dynamic instability was concomitantly suppressed. Consistently, MT ends in CSPP1-overexpressing cells were hyper-stabilized, while those in CSPP1-depleted cells were much more dynamic. This CSPP1-elicited stabilization of MTs was demonstrated to be achieved by suppressing intrinsic MT catastrophe and restricting the polymerization. Importantly, CSPP1-bound MTs were resistant to MCAK-mediated depolymerization. These findings delineate a previously uncharacterized CSPP1 activity that integrates MT end capping to orchestrate quiescent MTs.

PLK1 phosphorylation of ZW10 guides accurate chromosome segregation in mitosis.

Stable transmission of genetic information during cell division requires faithful chromosome segregation. Mounting evidence has demonstrated that PLK1 dynamics at kinetochores control correct kinetochore-microtubule attachments and subsequent silencing of the spindle checkpoint. However, the mechanisms underlying PLK1-mediated silencing of the spindle checkpoint remain elusive. Here, we identified a regulatory mechanism by which PLK1-elicited ZW10 phosphorylation regulates spindle checkpoint silencing in mitosis. ZW10 is a cognate substrate of PLK1, and the phosphorylation of ZW10 at Ser12 enables dynamic ZW10-Zwint1 interactions. Inhibition of ZW10 phosphorylation resulted in misaligned chromosomes, while persistent expression of phospho-mimicking ZW10 mutant caused premature anaphase, in which sister chromatids entangled as cells entered anaphase. These findings reveal the previously uncharacterized PLK1-ZW10 interaction through which dynamic phosphorylation of ZW10 fine-tunes accurate chromosome segregation in mitosis.

hUC-MSC-EV-miR-24 enhances the protective effect of dexmedetomidine preconditioning against myocardial ischemia-reperfusion injury through the KEAP1/Nrf2/HO-1 signaling.

The cardioprotective effect of microRNAs (miRNAs) on myocardial ischemic-reperfusion (I/R) injury has been documented. Here, we aim to decipher the mechanism of miR-24 delivered by human umbilical cord mesenchymal stem cell-derived extracellular vesicles (hUC-MSC-EVs) in myocardial I/R injury after dexmedetomidine (DEX) preconditioning. We collected and identified hUC-MSCs and extracted EVs, which were co-cultured with DEX-preconditioned hypoxia/reoxygenation (H/R) cardiomyocyte models or injected into I/R mouse models. The cardiomyocytes and myocardial injury were evaluated by molecular biology experiments. miR-24 was highly expressed in hUC-MSC-EVs. hUC-MSC-EVs could transfer miR-24 into cardiomyocytes where miR-24 augmented cell viability and inhibited cell apoptosis after DEX preconditioning. In the co-culture system of RAW264.7 macrophages with hUC-MSC-EVs, miR-24 promoted M2-type polarization of macrophages and reduced M1-type macrophage polarization. Mechanistically, miR-24 targeted KEAP1 and inhibited its expression, resulting in disruption of the Nrf2/HO-1 signaling. In vivo data confirmed that miR-24 delivered by hUC-MSC-EVs enhanced the suppressing effect of DEX preconditioning on inflammation and apoptosis in rats following myocardial I/R injury. Overall, miR-24 delivered by hUC-MSC-EVs can promote M2 polarization of macrophages and enhance the protective effect of DEX preconditioning on myocardial I/R injury by down-regulating the KEAP1/Nrf2/HO-1 signaling axis.

Catechol chitosan coated dual-loaded liposomes based on oxidation and saccharification mechanisms for enhancing skin anti-aging effects.

Skin aging has become a major urgent problem to be solved. Evidence reveals that oxidation and glycosylation are two dominant inducements of aging. Resveratrol (RES) with outstanding anti-oxidant effect and carnosine (CAR) with superb anti-glycation property were selected as two model drugs to evaluate the feasibility of their synergistic anti-aging effect. RES and CAR at the most desired mass ratio, supplying the most superior synergistic anti-aging effects were further encapsulated in liposomes (LP), which were separately coated with chitosan (CS) and catechol chitosan (Cat-CS) to increase the transdermal penetration. Their anti-aging efficacy was explored in human skin fibroblast (HSF) and human immortalized keratinocytes (HaCaT) cells, as well as the back skin of guinea pigs. Herein, RES and CAR at the mass ratio of 2:1 exhibited the most ideal synergistic anti-aging effect. The constructed liposomes have been shown to possess excellent fundamental properties and sustained-release properties. The aging-related indicator levels in the two cells and guinea pigs were obviously improved for the RES + CAR@Cat-CS-LP group. Additionally, skin appearance, tissue morphology, and collagen content were visibly improved, indicating its perfect anti-aging effect. In conclusion, RES + CAR@Cat-CS-LP is expected to be exploited as a potential anti-aging drug delivery system.

A target-triggered fluorescence-SERS dual-signal nano-system for real-time imaging of intracellular telomerase activity.

Telomerase (TE) is a promising diagnostic and prognostic biomarker for many cancers. Quantification of TE activity in living cells is of great significance in biomedical and clinical research. Conventional fluorescence-based sensors for quantification of intracellular TE may suffer from problems of fast photobleaching and auto-fluorescence of some endogenous molecules, and hence are liable to produce false negative or positive results. To address this issue, a fluorescence-SERS dual-signal nano-system for real-time imaging of intracellular TE was designed by functionalizing a bimetallic Au@Ag nanostructure with 4-p-mercaptobenzoic acid (internal standard SERS tag) and a DNA hybrid complex consisted of a telomerase primer strand and its partially complimentary strand modified with Rhodamine 6G. The bimetallic Au@Ag nanostructure serves as an excellent SERS-enhancing and fluorescence-quenching substrate. Intracellular TE will trigger the extension of the primer strand and cause the shedding of Rhodamine 6G-modified complimentary strand from the nano-system through intramolecular DNA strand displacement, resulting in the recovery of the fluorescence of Rhodamine 6G and decrease in its SERS signal. Both the fluorescence of R6G and the ratio between the SERS signals of 4-p-mercaptobenzoic acid and Rhodamine 6G can be used for in situ imaging of intracellular TE. Experimental results showed that the proposed nano-system was featured with low background, excellent cell internalization efficiency, good biocompatibility, high sensitivity, good selectivity, and robustness to false positive results. It can be used to distinguish cancer cells from normal ones, identify different types of cancer cells, as well as perform absolute quantification of intracellular TE, which endows it with great potential in clinical diagnosis, target therapy and prognosis of cancer patients.

Noninvasive Imaging of Tumor Glycolysis and Chemotherapeutic Resistance via De Novo Design of Molecular Afterglow Scaffold.

Chemotherapeutic resistance poses a significant challenge in cancer treatment, resulting in the reduced efficacy of standard chemotherapeutic agents. Abnormal metabolism, particularly increased anaerobic glycolysis, has been identified as a major contributing factor to chemotherapeutic resistance. To address this issue, noninvasive imaging techniques capable of visualizing tumor glycolysis are crucial. However, the currently available methods (such as PET, MRI, and fluorescence) possess limitations in terms of sensitivity, safety, dynamic imaging capability, and autofluorescence. Here, we present the de novo design of a unique afterglow molecular scaffold based on hemicyanine and rhodamine dyes, which holds promise for low-background optical imaging. In contrast to previous designs, this scaffold exhibits responsive "OFF-ON" afterglow signals through spirocyclization, thus enabling simultaneous control of photodynamic effects and luminescence efficacy. This leads to a larger dynamic range, broader detection range, higher signal enhancement ratio, and higher sensitivity. Furthermore, the integration of multiple functionalities simplifies probe design, eliminates the need for spectral overlap, and enhances reliability. Moreover, we have expanded the applications of this afterglow molecular scaffold by developing various probes for different molecular targets. Notably, we developed a water-soluble pH-responsive afterglow nanoprobe for visualizing glycolysis in living mice. This nanoprobe monitors the effects of glycolytic inhibitors or oxidative phosphorylation inhibitors on tumor glycolysis, providing a valuable tool for evaluating the tumor cell sensitivity to these inhibitors. Therefore, the new afterglow molecular scaffold presents a promising approach for understanding tumor metabolism, monitoring chemotherapeutic resistance, and guiding precision medicine in the future.

Real-time fluorescence imaging flow cytometry enabled by motion deblurring and deep learning algorithms.

Fluorescence imaging flow cytometry (IFC) has been demonstrated as a crucial biomedical technique for analyzing specific cell subpopulations from heterogeneous cellular populations. However, the high-speed flow of fluorescent cells leads to motion blur in cell images, making it challenging to identify cell types from the raw images. In this study, we present a real-time single-cell imaging and classification system based on a fluorescence microscope and deep learning algorithm, which is able to directly identify cell types from motion-blur images. To obtain annotated datasets of blurred images for deep learning model training, we developed a motion deblurring algorithm for the reconstruction of blur-free images. To demonstrate the ability of this system, deblurred images of HeLa cells with various fluorescent labels and HeLa cells at different cell cycle stages were acquired. The trained ResNet achieved a high accuracy of 96.6% for single-cell classification of HeLa cells in three different mitotic stages, with a short processing time of only 2 ms. This technology provides a simple way to realize single-cell fluorescence IFC and real-time cell classification, offering significant potential in various biological and medical applications.

Antagonizing the irreversible thrombomodulin-initiated proteolytic signaling alleviates age-related liver fibrosis via senescent cell killing.

Cellular senescence is a stress-induced, stable cell cycle arrest phenotype which generates a pro-inflammatory microenvironment, leading to chronic inflammation and age-associated diseases. Determining the fundamental molecular pathways driving senescence instead of apoptosis could enable the identification of senolytic agents to restore tissue homeostasis. Here, we identify thrombomodulin (THBD) signaling as a key molecular determinant of the senescent cell fate. Although normally restricted to endothelial cells, THBD is rapidly upregulated and maintained throughout all phases of the senescence program in aged mammalian tissues and in senescent cell models. Mechanistically, THBD activates a proteolytic feed-forward signaling pathway by stabilizing a multi-protein complex in early endosomes, thus forming a molecular basis for the irreversibility of the senescence program and ensuring senescent cell viability. Therapeutically, THBD signaling depletion or inhibition using vorapaxar, an FDA-approved drug, effectively ablates senescent cells and restores tissue homeostasis in liver fibrosis models. Collectively, these results uncover proteolytic THBD signaling as a conserved pro-survival pathway essential for senescent cell viability, thus providing a pharmacologically exploitable senolytic target for senescence-associated diseases.

Cognitive protection of sinomenine in type 2 diabetes mellitus through regulating the EGF/Nrf2/HO-1 signaling, the microbiota-gut-brain axis, and hippocampal neuron ferroptosis.

Recent years have witnessed a growing research interest in traditional Chinese medicine as a neuroprotective nutrient in the management of diabetic cognitive dysfunction. However, the underlying molecular mechanisms of sinomenine in mediating ferroptosis of hippocampal neurons have been poorly understood. This study sought to decipher the potential effect and molecular mechanism of sinomenine in the cognitive dysfunction following type 2 diabetes mellitus (T2DM). Multi-omics analysis was conducted to identify the microbiota-gut-brain axis in T2DM patient samples obtained from the publicly available database. In HT-22 cells, erastin was utilized to create a ferroptosis model, and streptozotocin was injected intraperitoneally to create a rat model of DM. It was noted that intestinal flora imbalance occurred in patients with T2DM-associated cognitive dysfunction. Sinomenine could reduce Erastin-induced hippocampus neuronal ferroptosis by increasing EGF expression. EGF protected hippocampal neurons against ferroptosis by activating the Nrf2/HO-1 signaling pathway. Furthermore, in vivo results confirmed that sinomenine blocked ferroptosis of hippocampal neurons and alleviated cognitive dysfunction in T2DM rats. Collectively, these results suggest that sinomenine confers neuroprotective effects by curtailing hippocampal neuron ferroptosis via the EGF/Nrf2/HO-1 signaling and microbiota-gut-brain axis. It may be a candidate for the treatment of diabetic cognitive dysfunction.

Cardioprotective effects of circ_0002612 in myocardial ischemia/reperfusion injury correlate with disruption of miR-30a-5p-dependent Ppargc1a inhibition.

INTRODUCTION: Novel mechanistic insights into the effects of circular RNAs (circRNAs) on the physiology and pathology of cardiovascular diseases are under increasingly active investigation. This study defined the cardioprotective role and mechanistic actions of circ_0002612 in myocardial ischemia/reperfusion injury (MI/RI). METHODS: MI/RI was induced in mice by ligation of the left anterior descending (LAD) artery followed by reperfusion, and the in vitro model was established in cultured cardiomyocytes under hypoxia/reoxygenation (H/R) conditions. Interaction among circ_0002612, miR-30a-5p, Ppargc1a, and NLRP3 was predicted by bioinformatics analysis and further experimentally identified. Gain- and loss-of-function experiments were performed to evaluate the effect of the circ_0002612/miR-30a-5p/Ppargc1a/NLRP3 axis on the cardiac function and myocardial infarction of I/R-injured mice, as well as viability and apoptosis of H/R-challenged cardiomyocytes. RESULTS: In the myocardial tissues of MI/RI mice, miR-30a-5p was negatively correlated with circ_0002612 or Ppargc1a, but circ_0002612 was positively correlated with the expression of Ppargc1a. circ_0002612 competitively bound to miR-30a-5p to release expression of its target gene Ppargc1a. circ_0002612 promoted cardiomyocyte viability while suppressing the apoptosis by impairing the miR-30a-5p-mediated inhibition of Ppargc1a. Additionally, Ppargc1a inhibited the expression of NLRP3 and consequently facilitated cardiomyocyte proliferation while suppressing cell apoptosis. By inhibiting the expression of NLRP3, circ_0002612 protected mice from MI/RI. CONCLUSION: Overall, this study demonstrates the cardioprotective role of circ_0002612 against MI/RI, which may be a viable target for MI/RI.

Delivery of miR-130a-3p Through Adipose-Derived Stem Cell-Secreted EVs Protects Against Diabetic Peripheral Neuropathy via DNMT1/NRF2/HIF1α/ACTA1 Axis.

Peripheral neuropathy is common in diabetic patients and can lead to amputations or foot ulcers. microRNAs (miRNAs) possess crucial roles in diabetic peripheral neuropathy (DPN). This study aims to investigate the role miR-130a-3p played in DPN and its underlying molecular mechanisms. miR-130a-3p expression in clinical tissue samples, established DPN rat models, and extracellular vesicles (EVs) derived from adipose-derived stem cells (ADSCs) were determined. Schwann cells (SCs) were co-cultured with ADSC-derived EVs and treated with high glucose. The direct relationship and functional significance of miR-130a-3p, DNMT1, nuclear factor E2-related factor 2 (NRF2), hypoxia-inducible factor-1α (HIF1α), and skeletal muscle actin alpha 1 (ACTA1) was identified. The in vitro and in vivo implication of ADSC-derived EVs carrying miR-130a-3p was assessed. miR-130a-3p was poorly expressed in DPN patients and rats but highly expressed in ADSC-derived EVs. miR-130a-3p could be delivered to SCs through ADSC-derived EVs to inhibit SC apoptosis and promote proliferation under a high-glucose environment. miR-130a-3p activated NRF2/HIF1α/ACTA1 axis through down-regulating DNMT1. In vivo injection of ADSC-derived EVs activated NRF2/HIF1α/ACTA11 axis to promote angiogenesis in DPN rat model. These data together supported that ADSC-derived EVs carrying miR-130a-3p could alleviate DPN by accelerating SC proliferation and inhibiting apoptosis, providing a potential treatment against DPN.