Optogenetic manipulation of lysosomal physiology and autophagy-dependent clearance of amyloid beta.

Lysosomes are degradation centers of cells and intracellular hubs of signal transduction, nutrient sensing, and autophagy regulation. Dysfunction of lysosomes contributes to a variety of diseases, such as lysosomal storage diseases (LSDs) and neurodegeneration, but the mechanisms are not well understood. Altering lysosomal activity and examining its impact on the occurrence and development of disease is an important strategy for studying lysosome-related diseases. However, methods to dynamically regulate lysosomal function in living cells or animals are still lacking. Here, we constructed lysosome-localized optogenetic actuators, named lyso-NpHR3.0, lyso-ArchT, and lyso-ChR2, to achieve optogenetic manipulation of lysosomes. These new actuators enable light-dependent control of lysosomal membrane potential, pH, hydrolase activity, degradation, and Ca2+ dynamics in living cells. Notably, lyso-ChR2 activation induces autophagy through the mTOR pathway, promotes Aß clearance in an autophagy-dependent manner in cellular models, and alleviates Aß-induced paralysis in the Caenorhabditis elegans model of Alzheimer's disease. Our lysosomal optogenetic actuators supplement the optogenetic toolbox and provide a method to dynamically regulate lysosomal physiology and function in living cells and animals.

Lysosomal cation channel TRPML1 suppression sensitizes acute myeloid leukemia cells to chemotherapeutics by inhibiting autophagy.

Despite the implementation of novel therapeutic regimens and extensive research efforts, chemoresistance remains a formidable challenge in the treatment of acute myeloid leukemia (AML). Notably, the involvement of lysosomes in chemoresistance has sparked interest in developing lysosome-targeted therapies to sensitize tumor cells to currently approved chemotherapy or as innovative pharmacological approaches. Moreover, as ion channels on the lysosomal membrane are critical regulators of lysosomal function, they present potential as novel targets for enhancing chemosensitivity. Here, we discovered that the expression of a lysosomal cation channel, namely transient receptor potential mucolipin 1 (TRPML1), was elevated in AML cells. Inhibiting TRPML1 individually does not impact the proliferation and apoptosis of AML cells. Importantly, inhibition of TRPML1 demonstrated the potential to modulate the sensitivity of AML cells to chemotherapeutic agents. Exploration of the underlying mechanisms revealed that suppression of TRPML1 impaired autophagy while concurrently increasing the production of reactive oxygen species (ROS) and ROS-mediated lipid peroxidation (Lipid-ROS) in AML cells. Finally, the knockdown of TRPML1 significantly reduced OCI-AML3 tumor growth following chemotherapy in a mouse model of human leukemia. In summary, targeting TRPML1 represents a promising approach for combination therapy aimed at enhancing chemosensitivity in treating AML.

Lysosomal K+ channel TMEM175 promotes apoptosis and aggravates symptoms of Parkinson's disease.

Lysosomes are degradative organelles and play vital roles in a variety of cellular processes. Ion channels on the lysosomal membrane are key regulators of lysosomal function. TMEM175 has been identified as a lysosomal potassium channel, but its modulation and physiological functions remain unclear. Here, we show that the apoptotic regulator Bcl-2 binds to and inhibits TMEM175 activity. Accordingly, Bcl-2 inhibitors activate the channel in a caspase-independent way. Increased TMEM175 function inhibits mitophagy, disrupts mitochondrial homeostasis, and increases production of reactive oxygen species (ROS). ROS further activates TMEM175 and thus forms a positive feedback loop to augment apoptosis. In a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease (PD), knockout (KO) of TMEM175 mitigated motor impairment and dopaminergic (DA) neuron loss, suggesting that TMEM175-mediated apoptosis plays an important role in Parkinson's disease (PD). Overall, our study reveals that TMEM175 is an important regulatory site in the apoptotic signaling pathway and a potential therapeutic target for Parkinson's disease (PD).

Aptamer-Based Detection of Circulating Targets for Precision Medicine.

The past decade has witnessed ongoing progress in precision medicine to improve human health. As an emerging diagnostic technique, liquid biopsy can provide real-time, comprehensive, dynamic physiological and pathological information in a noninvasive manner, opening a new window for precision medicine. Liquid biopsy depends on the sensitive and reliable detection of circulating targets (e.g., cells, extracellular vesicles, proteins, microRNAs) from body fluids, the performance of which is largely governed by recognition ligands. Aptamers are single-stranded functional oligonucleotides, capable of folding into unique tertiary structures to bind to their targets with superior specificity and affinity. Their mature evolution procedure, facile modification, and affinity regulation, as well as versatile structural design and engineering, make aptamers ideal recognition ligands for liquid biopsy. In this review, we present a broad overview of aptamer-based liquid biopsy techniques for precision medicine. We begin with recent advances in aptamer selection, followed by a summary of state-of-the-art strategies for multivalent aptamer assembly and aptamer interface modification. We will further describe aptamer-based micro-/nanoisolation platforms, aptamer-enabled release methods, and aptamer-assisted signal amplification and detection strategies. Finally, we present our perspectives regarding the opportunities and challenges of aptamer-based liquid biopsy for precision medicine.

Aptamer-LYTACs for Targeted Degradation of Extracellular and Membrane Proteins.

Recently, lysosome targeting chimeras (LYTACs) have emerged as a promising technology that expands the scope of targeted protein degradation to extracellular targets. However, the preparation of chimeras by conjugation of the antibody and trivalent N-acetylgalactosamine (tri-GalNAc) is a complex and time-consuming process. The large uncertainty in number and position and the large molecular weights of the chimeras result in low internalization efficiency. To circumvent these problems, we developed the first aptamer-based LYTAC (Apt-LYTAC) to realize liver-cell-specific degradation of extracellular and membrane proteins by conjugating aptamers to tri-GalNAc. Taking advantage of the facile synthesis and low molecular weight of the aptamer, the Apt-LYTACs can efficiently and quickly degrade the extracellular protein PDGF and the membrane protein PTK7 through a lysosomal degradation pathway. We anticipate that the novel Apt-LYTACs will expand the usage of aptamers and provide a new dimension for targeted protein degradation.

In situ Raman enhancement strategy for highly sensitive and quantitative lateral flow assay.

As a paper-based analytical platform, lateral flow assay (LFA) gets benefit from the rapid analysis, low cost, high selectivity, good stability, and user-friendliness, and thus has been widely used in rapid screening or assisted diagnosis. Nevertheless, LFA still suffers from low detection sensitivity via the naked eye, limiting its applications to qualitative and semi-quantitative tests. To enhance the signal readout, various nanoparticle signal tags have been employed to replace traditional colloidal gold nanoparticles (AuNPs), such as fluorescent nanoparticles (FNPs), magnetic nanoparticles (MNPs), and Raman reporter-labeled nanoparticles. In particular, Raman reporter-labeled nanoparticles are extremely sensitive due to remarkable signal enhancement effect on metal surface. However, the application of LFA is still hampered by the poor stability of Raman reporter-labeled nanoparticles. Herein, we developed an in situ Raman enhancement strategy to create a surface-enhanced Raman scattering (SERS) signal on the AuNPs, shortened as "i-SERS," which not only preserves the original advantages of the colloidal gold strip (AuNPs-LFA), but also realizes highly sensitive and quantitative detection. We applied the i-SERS for procalcitonin (PCT) detection. The experimental process takes only 16 min, and the limit of detection (LOD) is 0.03 ng mL-1, far below the value using AuNPs-LFA. These results indicate that i-SERS assay was highly sensitive and suitable for the rapid detection of PCT.

Recent Advances in Exosomal miRNA Biosensing for Liquid Biopsy.

As a noninvasive detection technique, liquid biopsy plays a valuable role in cancer diagnosis, disease monitoring, and prognostic assessment. In liquid biopsies, exosomes are considered among the potential biomarkers because they are important bioinformation carriers for intercellular communication. Exosomes transport miRNAs and, thus, play an important role in the regulation of cell growth and function; therefore, detection of cancer cell-derived exosomal miRNAs (exo-miRNAs) gives effective information in liquid biopsy. The development of sensitive, convenient, and reliable exo-miRNA assays will provide new perspectives for medical diagnosis. This review presents different designs and detection strategies of recent exo-miRNA assays in terms of signal transduction and amplification, as well as signal detection. In addition, this review outlines the current attempts at bioassay methods in liquid biopsies. Lastly, the challenges and prospects of exosome bioassays are also considered.

HUNTER-Chip: Bioinspired Hierarchically Aptamer Structure-Based Circulating Fetal Cell Isolation for Non-Invasive Prenatal Testing.

Isolation and genetic analysis of circulating fetal cells from billions of maternal cells in peripheral blood are the cornerstone of fetal cell-based non-invasive prenatal testing. Inspired by the hierarchically multivalent architecture for enhanced capture of nature, an aptamer-based Hierarchically mUltivalent aNTibody mimic intERface (HUNTER) was designed with a tremendous avidity effect for highly efficient capture and non-destructive release of fetal cells. It was engineered by grafting Y-shaped DNA nanostructures to a linear polymer chain, creating a flexible polymer chain with bivalent aptamer side chains. This hierarchical arrangement of the aptamer ensures morphological complementarity, collective multiple-site interaction, and multivalent recognition between the aptamer and target cells. In combination with a deterministic lateral displacement (DLD)-patterned microdevice named as HUNTER-Chip, it achieves a binding affinity over 65-fold and a capture efficiency over 260%-fold due to the combination of hierarchically designed aptamers and frequent cell-ligand collision created by DLD. Moreover, a nuclease-assisted cell release strategy facilitates the release of fetal cells for gene analysis, such as fluorescence in situ hybridization. With the advantages of high affinity, excellent capture efficiency, and compatible downstream analysis, the HUNTER-Chip holds great potential for non-invasive prenatal diagnosis.

Coupling Aptamer-based Protein Tagging with Metabolic Glycan Labeling for In Situ Visualization and Biological Function Study of Exosomal Protein-Specific Glycosylation.

Exosomal glycoproteins play important roles in many physiological and pathological functions. Herein, we developed a dual labeling strategy based on a protein-specific aptamer tagging and metabolic glycan labeling for visualizing glycosylation of specific proteins on exosomes. The glycosylation of exosomal PD-L1 (exoPD-L1) was imaged in situ using intramolecular fluorescence resonance energy transfer (FRET) between fluorescent PD-L1 aptamers bound on exoPD-L1 and fluorescent tags on glycans introduced via metabolic glycan labeling. This method enables in situ visualization and biological function study of exosomal protein glycosylation. Exosomal PD-L1 glycosylation was confirmed to be required in interaction with PD-1 and participated in inhibiting of CD8+ T cell proliferation. This is an efficient and non-destructive method to study the presence and function of exosomal protein-specific glycosylation in situ, which provides a powerful tool for exosomal glycoproteomics research.

Tracing Tumor-Derived Exosomal PD-L1 by Dual-Aptamer Activated Proximity-Induced Droplet Digital PCR.

Tumor-derived exosomal proteins have emerged as promising biomarkers for cancer diagnosis, but the quantitation accuracy is hindered by large numbers of normal cell-derived exosomes. Herein, we developed a dual-target-specific aptamer recognition activated in situ connection system on exosome membrane combined with droplet digital PCR (ddPCR) (TRACER) for quantitation of tumor-derived exosomal PD-L1 (Exo-PD-L1 ). Leveraging the high binding affinity of aptamers, excellent selectivity of dual-aptamer recognition, and the high sensitivity of ddPCR, this method exhibits significant sensitivity and selectivity for tracing tumor-derived Exo-PD-L1 in a wash-free manner. Due to the excellent sensitivity, the level of tumor-derived Exo-PD-L1 detected by TRACER can distinguish cancer patients from healthy donors, and for the first time was identified as a more reliable tumor diagnostic marker than total Exo-PD-L1 . The TRACER strategy holds great potential for converting exosomes into reliable clinical indicators and exploring the biological functions of exosomes.

Activation of Aptamers with Gain of Function by Small-Molecule-Clipping of Intramolecular Motifs.

Click reactions have the advantages of high reactivity, excellent orthogonality, and synthetic accessibility. Inspired by click reactions, we propose the concept of "clipped aptamers", whose binding affinity is regulated by the "clip"-like specific interaction between a synthetic DNA-mismatch-binding small molecule (molecular glue, Z-NCTS) and the preset CGG/CGG sequences in nucleic acid sequences. In this study, we investigated a Z-NCTS-mediated de novo selection of clipped aptamers against epithelial cell adhesion molecule. The generated clipped aptamers can achieve the efficient transition from a binding-inactive state to an active state by clipping of Z-NCTS with two CGG sites, which otherwise would not hybridize. The experimental and simulation results showed that the clipped aptamer had ideal binding thermodynamics and the ability to regulate cellular adhesion. Because of this superior activated mechanism and structural diversity, clipped aptamers hold great potential in biosensing, imaging, conditional gene- and cellular behavior-regulation, and drug delivery.

Discovery of Aptamers Targeting the Receptor-Binding Domain of the SARS-CoV-2 Spike Glycoprotein.

The World Health Organization has declared the outbreak of a novel coronavirus (SARS-CoV-2 or 2019-nCoV) as a global pandemic. However, the mechanisms behind the coronavirus infection are not yet fully understood, nor are there any targeted treatments or vaccines. In this study, we identified high-binding-affinity aptamers targeting SARS-CoV-2 RBD, using an ACE2 competition-based aptamer selection strategy and a machine learning screening algorithm. The Kd values of the optimized CoV2-RBD-1C and CoV2-RBD-4C aptamers against RBD were 5.8 nM and 19.9 nM, respectively. Simulated interaction modeling, along with competitive experiments, suggests that two aptamers may have partially identical binding sites at ACE2 on SARS-CoV-2 RBD. These aptamers present an opportunity for generating new probes for recognition of SARS-CoV-2 and could provide assistance in the diagnosis and treatment of SARS-CoV-2 while providing a new tool for in-depth study of the mechanisms behind the coronavirus infection.

DNA Nanolithography Enables a Highly Ordered Recognition Interface in a Microfluidic Chip for the Efficient Capture and Release of Circulating Tumor Cells.

Microfluidic chips with nano-scale structures have shown great potential, but the fabrication and cost issues restrict their application. Herein, we propose a conceptually new "DNA nanolithography in a microfluidic chip" by using sub-10 nm three-dimensional DNA structures (TDNs) as frameworks with a pendant aptamer at the top vertex (ApTDN-Chip). The nano-scale framework ensures that the aptamer is in a highly ordered upright orientation, avoiding the undesired orientation or crowding effects caused by conventional microfluidic interface fabrication processes. Compared with a monovalent aptamer modified chip, the capture efficiency of ApTDN-Chip was enhanced nearly 60 % due to the highly precise dimension and rigid framework of TDNs. In addition, the scaffolds make DNase I more accessible to the aptamer with up to 83 % release efficiency and 91 % cell viability, which is fully compatible with downstream molecular analysis. Overall, this strategy provides a novel perspective on engineering nano-scaffolds to achieve a more ordered nano-topography of microfluidic chips.

Bacterial Extracellular Electron Transfer Occurs in Mammalian Gut.

As a well-studied biochemical reduction process in environmental microbiology, extracellular electron transfer (EET) was recently discovered in bacteria closely related to human health, and orthologues of a flavin-based EET gene were found in the genomes of many species across Firmicutes, a major phylum in mammalian gut microbiota. However, EET has not yet been confirmed to occur in mammalian gut, the presence of which may have broad physiological influences. Toward this end, here we first confirmed the occurrence of EET in mouse gut microbiotas cultured in vitro. Cyclic voltammetry analysis was then performed by directly inserting electrodes into the mouse cecum under anaerobic conditions, and a characteristic catalytic wave was observed in the gut of conventional but not germ-free mouse, proving the existence of in vivo bacterial EET. We also detected similar catalytic waves in the cecal microbiotas of rat and guinea pig in vivo, suggesting EET's high prevalence in mammalian intestines. Our finding on the bacterial electron production in mammalian guts offers a new bioelectrochemical scope for deciphering the complex microbiology of gut bacteria and its effects on host physiology.

Surface-Enhanced Raman Scattering Active Plasmonic Nanoparticles with Ultrasmall Interior Nanogap for Multiplex Quantitative Detection and Cancer Cell Imaging.

Due to its large enhancement effect, nanostructure-based surface-enhanced Raman scattering (SERS) technology had been widely applied for bioanalysis and cell imaging. However, most SERS nanostructures suffer from poor signal reproducibility, which hinders the application of SERS nanostructures in quantitative detection. We report an etching-assisted approach to synthesize SERS-active plasmonic nanoparticles with 1 nm interior nanogap for multiplex quantitative detection and cancer cell imaging. Raman dyes and methoxy poly(ethylene glycol) thiol (mPEG-SH) were attached to gold nanoparticles (AuNPs) to prepare gold cores. Next, Ag atoms were deposited on gold cores in the presence of Pluronic F127 to form a Ag shell. HAuCl4 was used to etch the Ag shell and form an interior nanogap in Au@AgAuNPs, leading to increased Raman intensity of dyes. SERS intensity distribution of Au@AgAuNPs was found to be more uniform than that of aggregated AuNPs. Finally, Au@AgAuNPs were used for multiplex quantitative detection and cancer cell imaging. With the advantages of simple and rapid preparation of Au@AgAuNPs with highly uniform, stable, and reproducible Raman intensity, the method reported here will widen the applications of SERS-active nanoparticles in diagnostics and imaging.

DNA-Mediated Morphological Control of Silver Nanoparticles.

It is demonstrated that DNA can be used to control the synthesis of silver nanoplates with different morphologies using spherical silver seeds. UV-vis spectroscopy, transmission electron microscopy, scanning electron microscopy, X-ray photoelectron spectroscopy, and Raman spectroscopy are used to characterize the synthesized nanoparticles. Silver nanoprisms are encoded by poly C and poly G, while silver flower bouquets and silver nanodiscs are synthesized using poly A and poly T, respectively. The length of DNA is found to have little effect on the morphology of silver nanoparticles. Moreover, the synthesized silver nanoplates are found to have high surface enhanced Raman scattering enhancement ability, good antibacterial activity, and good biocompatibility. These discoveries will broaden the application of DNA in nanoscience and will provide a new platform to investigate the interaction between DNA sequences and silver nanoparticles.

Locus-specific detection of pseudouridine with CRISPR-Cas13a.

We combined the CRISPR-Cas13a system with CMC chemical labeling, developing an approach that enables precise identification of pseudouridine (Ψ) sites at specific loci within ribosomal RNA (rRNA), messenger RNA (mRNA) and small nuclear RNAs (snRNA). This method, with good efficiency and simplicity, detects Ψ sites through fluorescence measurement, providing a straightforward and fast validation for targeted Ψ sites of interest.

Correctable Landmark Discovery Via Large Models for Vision-Language Navigation.

Vision-Language Navigation (VLN) requires the agent to follow language instructions to reach a target position. A key factor for successful navigation is to align the landmarks implied in the instruction with diverse visual observations. However, previous VLN agents fail to perform accurate modality alignment especially in unexplored scenes, since they learn from limited navigation data and lack sufficient open-world alignment knowledge. In this work, we propose a new VLN paradigm, called COrrectable LaNdmark DiScOvery via Large ModEls (CONSOLE). In CONSOLE, we cast VLN as an open-world sequential landmark discovery problem, by introducing a novel correctable landmark discovery scheme based on two large models ChatGPT and CLIP. Specifically, we use ChatGPT to provide rich open-world landmark cooccurrence commonsense, and conduct CLIP-driven landmark discovery based on these commonsense priors. To mitigate the noise in the priors due to the lack of visual constraints, we introduce a learnable cooccurrence scoring module, which corrects the importance of each cooccurrence according to actual observations for accurate landmark discovery. We further design an observation enhancement strategy for an elegant combination of our framework with different VLN agents, where we utilize the corrected landmark features to obtain enhanced observation features for action decision. Extensive experimental results on multiple popular VLN benchmarks (R2R, REVERIE, R4R, RxR) show the significant superiority of CONSOLE over strong baselines. Especially, our CONSOLE establishes the new state-of-the-art results on R2R and R4R in unseen scenarios.

Engineered aptamers for molecular imaging.

Molecular imaging, including quantification and molecular interaction studies, plays a crucial role in visualizing and analysing molecular events occurring within cells or organisms, thus facilitating the understanding of biological processes. Moreover, molecular imaging offers promising applications for early disease diagnosis and therapeutic evaluation. Aptamers are oligonucleotides that can recognize targets with a high affinity and specificity by folding themselves into various three-dimensional structures, thus serving as ideal molecular recognition elements in molecular imaging. This review summarizes the commonly employed aptamers in molecular imaging and outlines the prevalent design approaches for their applications. Furthermore, it highlights the successful application of aptamers to a wide range of targets and imaging modalities. Finally, the review concludes with a forward-looking perspective on future advancements in aptamer-based molecular imaging.