High-fidelity ATP imaging via an isothermal cascade catalytic amplifier.

Artificial catalytic DNA circuits that can identify, transduce and amplify the biomolecule of interest have supplemented a powerful toolkit for visualizing various biomolecules in cancer cells. However, the non-specific response in normal tissues and the low abundance of analytes hamper their extensive biosensing and biomedicine applications. Herein, by combining tumor-responsive MnO2 nanoparticles with a specific stimuli-activated cascade DNA amplifier, we propose a multiply guaranteed and amplified ATP-sensing platform via the successive cancer-selective probe exposure and stimulation procedures. Initially, the GSH-degradable MnO2 nanocarrier, acting as a tumor-activating module, ensures the accurate delivery of the cascade DNA amplifier into GSH-rich cancer cells and simultaneously provides adequate Mn2+ cofactors for facilitating the DNAzyme biocatalysis. Then, the released cascade amplifier, acting as an ATP-monitoring module, fulfills the precise and sensitive analysis of low-abundance ATP in cancer cells where the catalyzed hairpin assembly (CHA) is integrated with the DNAzyme biocatalyst for higher signal gain. Additionally, the cascade catalytic amplifier achieved tumor-specific activated photodynamic therapy (PDT) after integrating an activatable photosensitizer into the system. This homogeneous cascade catalytic aptasensing circuit can detect low-abundance endogenous ATP of cancer cells, due to its intrinsically rich recognition repertoire and avalanche-mimicking hierarchical acceleration, thus demonstrating broad prospects for analyzing clinically important biomolecules and the associated physiological processes.

Airway basal cells show regionally distinct potential to undergo metaplastic differentiation.

Basal cells are multipotent stem cells of a variety of organs, including the respiratory tract, where they are major components of the airway epithelium. However, it remains unclear how diverse basal cells are and how distinct subpopulations respond to airway challenges. Using single cell RNA-sequencing and functional approaches, we report a significant and previously underappreciated degree of heterogeneity in the basal cell pool, leading to identification of six subpopulations in the adult murine trachea. Among these, we found two major subpopulations, collectively comprising the most uncommitted of all the pools, but with distinct gene expression signatures. Notably, these occupy distinct ventral and dorsal tracheal niches and differ in their ability to self-renew and initiate a program of differentiation in response to environmental perturbations in primary cultures and in mouse injury models in vivo. We found that such heterogeneity is acquired prenatally, when the basal cell pool and local niches are still being established, and depends on the integrity of these niches, as supported by the altered basal cell phenotype of tracheal cartilage-deficient mouse mutants. Finally, we show that features that distinguish these progenitor subpopulations in murine airways are conserved in humans. Together, the data provide novel insights into the origin and impact of basal cell heterogeneity on the establishment of regionally distinct responses of the airway epithelium during injury-repair and in disease conditions.

A Mitochondrial Oxidative Stress Amplifier to Overcome Hypoxia Resistance for Enhanced Photodynamic Therapy.

Hypoxia-induced resistance to tumor treatment restricts further development of photodynamic therapy. Instead of simple reoxygenation to relieve hypoxia in traditional therapeutic approaches, a mitochondria-targeted reactive oxygen species (ROS) amplifier is constructed to reverse hypoxia resistance and enhance tumor sensitivity to hypoxia-resistant photodynamic therapy. Mesoporous silica nanoparticles are modified with triphenylphosphine to enhance its blood circulation and endow it with mitochondria-targeted specificity. α-Tocopherol succinate and indocyanine green are loaded in mitochondria-targeted mesoporous silica nanoparticles to reduce innate oxygen consumption by blocking mitochondrial respiration chain, leading to endogenous mitochondrial ROS burst and imaging-guided photodynamic therapy. This mitochondria-targeted oxidative stress amplifier not only disrupts mitochondrial redox homeostasis and triggers long-term high oxidative stress but also makes tumor more sensitive to hypoxia-resistant photodynamic therapy. The imaging-guided ROS amplifier confirms the feasibility and effectiveness of both in vitro and in vivo anticancer performance, suggesting a promising clinical strategy in hypoxia-related tumor treatment.

A Cooperatively Activatable DNA Nanoprobe for Cancer Cell-Selective Imaging of ATP.

DNA-based nanoprobes have attracted extensive interest in the field of bioanalysis. Notably, engineered DNA nanoprobes that can respond to multiple pathological parameters are desirable to detect targets precisely. Here we design a split aptamer/DNAzyme (aptazyme)-based DNA probe for fluorescence detection of ATP and further develop a cooperatively activatable DNA nanoprobe for tumor-specific imaging of ATP in vivo. The DNA nanoprobes comprising split aptazyme-coated MnO2 nanovectors have high stability and are synergistically activated by multiple biomarkers, GSH and ATP. Upon stimuli by overexpressed GSH in tumor cells, this DNA nanoprobe can release the aptazyme and self-supply cofactor Mn2+ of the DNAzyme. Sequentially, intracellular ATP induces the proper folding of the split ATP aptamer and Mn2+-dependent DNAzyme, which activates the specific cleavage of substrate and generates the optical readout signal. This nanoprobe exhibits remarkable resistance to enzymatic degradation, satisfactory biosafety, identifies ATP specifically within cancer cells, and selectively lights up solid tumors. Our research provides a reliable method for ATP imaging in cancer cells and opens a new avenue for biochemical research and highly accurate disease diagnosis.

Cascaded Amplifier Nanoreactor for Efficient Photodynamic Therapy.

Photodynamic therapy (PDT) utilizes reactive oxygen species (ROS) to treat established diseases and has attracted growing attention in the field of cancer therapy. However, in a tumor microenvironment (TME), the inherent hypoxia and high level of antioxidants severely hamper the efficacy of ROS generation. Here, we describe a cascaded amplifier nanoreactor based on self-assembled nanofusiforms for persistent oxygenation to amplify ROS levels. The nanofusiform assembly is capable of photothermal and photodynamic treatment and regulation of redox oxidation stress by antioxidant depletion to prevent ROS tolerance. The Pt nanozyme decoration of the nanofusiform enables efficient oxygen supplements via Pt nanozyme-catalyzed decomposition of H2O2 overexpressed in TME and generation of O2. Furthermore, the temperature elevation resulted from the photothermal effect of the nanofusiform increases the catalase-like catalytic activity of the Pt nanozyme for boosted oxygen generation. Thus, such a triple cascade strategy using nanozyme-based nanofusiforms amplifies the ROS level by continuous oxygenation, enhancing the efficacy of PDT in vitro and in vivo. Meanwhile, an in vivo multi-modal imaging including near-infrared fluorescence imaging, photothermal imaging, and magnetic resonance imaging achieves precise tumor diagnosis. The rationally designed nanofusiform acts as an efficient ROS amplifier through multidimension strengthening of continuous oxygenation, providing a potential smart nanodrug for cancer therapy.

Distinct and sequential re-replication barriers ensure precise genome duplication.

Achieving complete and precise genome duplication requires that each genomic segment be replicated only once per cell division cycle. Protecting large eukaryotic genomes from re-replication requires an overlapping set of molecular mechanisms that prevent the first DNA replication step, the DNA loading of MCM helicase complexes to license replication origins, after S phase begins. Previous reports have defined many such origin licensing inhibition mechanisms, but the temporal relationships among them are not clear, particularly with respect to preventing re-replication in G2 and M phases. Using a combination of mutagenesis, biochemistry, and single cell analyses in human cells, we define a new mechanism that prevents re-replication through hyperphosphorylation of the essential MCM loading protein, Cdt1. We demonstrate that Cyclin A/CDK1 can hyperphosphorylate Cdt1 to inhibit MCM re-loading in G2 phase. The mechanism of inhibition is to block Cdt1 binding to MCM independently of other known Cdt1 inactivation mechanisms such as Cdt1 degradation during S phase or Geminin binding. Moreover, our findings suggest that Cdt1 dephosphorylation at the mitosis-to-G1 phase transition re-activates Cdt1. We propose that multiple distinct, non-redundant licensing inhibition mechanisms act in a series of sequential relays through each cell cycle phase to ensure precise genome duplication.

Programming DNA Nanoassembly for Enhanced Photodynamic Therapy.

Photodynamic therapy (PDT) has extraordinary promise for the treatment of many cancers. However, its clinical progress is impaired by the intrinsic hypoxic tumor microenvironment that limits PDT efficacy and the safety concern associated with biological specificity of photosensitizers or vehicles. Now it is demonstrated that rationally designed DNA nanosponges can load and delivery photosensitizer effectively, target tumor precisely, and relieve hypoxia-associated resistance remarkably to enhance the efficacy of PDT. Specifically, the approach exhibits a facile assembly process, provides programmable and versatile nanocarriers, and enables robust in vitro and in vivo anti-cancer efficacy with excellent biosafety. These findings represent a practical and safe approach by designer DNA nanoassemblies to combat cancer effectively and suggest a powerful strategy for broad biomedical application of PDT.

Cdt1 stabilizes kinetochore-microtubule attachments via an Aurora B kinase-dependent mechanism.

Robust kinetochore-microtubule (kMT) attachment is critical for accurate chromosome segregation. G2/M-specific depletion of human Cdt1 that localizes to kinetochores in an Ndc80 complex-dependent manner leads to abnormal kMT attachments and mitotic arrest. This indicates an independent mitotic role for Cdt1 in addition to its prototypic function in DNA replication origin licensing. Here, we show that Cdt1 directly binds to microtubules (MTs). Endogenous or transiently expressed Cdt1 localizes to both mitotic spindle MTs and kinetochores. Deletion mapping of Cdt1 revealed that the regions comprising the middle and C-terminal winged-helix domains but lacking the N-terminal unstructured region were required for efficient MT binding. Mitotic kinase Aurora B interacts with and phosphorylates Cdt1. Aurora B-phosphomimetic Cdt1 exhibited attenuated MT binding, and its cellular expression induced defective kMT attachments with a concomitant delay in mitotic progression. Thus we provide mechanistic insight into how Cdt1 affects overall kMT stability in an Aurora B kinase phosphorylation-dependent manner; which is envisioned to augment the MT-binding of the Ndc80 complex.

Nanog suppresses cell migration by downregulating thymosin ß4 and Rnd3.

Nanog, Sox2, and Oct4 are key transcription factors critical for the pluripotency and self-renewal of embryonic stem (ES) cells. Their downregulations lead to differentiation, accompanied with changes in cell motility. Whether these factors impact cell motility directly, however, is not clear. Here we addressed this question by initially assessing their effect in non-stem cells. We found that the ectopic expression of Nanog, Sox2, or Oct4 markedly inhibited ECV304 cell migration. Detailed examinations revealed that Nanog induced disorganizations of the actin cytoskeleton and peripheral localizations of focal adhesions. These effects required its DNA-binding domain and are thus transcription dependent. Furthermore, thymosin ß4 and Rnd3 were identified as its downstream targets. Their depletions in ECV304 cells by RNAi phenocopied the ectopic expression of Nanog in both cell motility and actin organization, whereas their ectopic expressions rescued the migration defect of Nanog overexpression. Both proteins were upregulated during mouse ES cell differentiation. Their levels in the pluripotent mouse P19 cells also increased upon Nanog ablation, coincident with an increase in cell motility. Moreover, persistent expression of Nanog in zebrafish embryos suppressed gastrulation and cell migration. These results indeed suggest a dual role of certain transcription factors in the orchestration of differentiation and motility.

miR-129-3p controls cilia assembly by regulating CP110 and actin dynamics.

Ciliogenesis requires the removal of CP110 from the mother centriole; actin dynamics also influence ciliation, at least partly by affecting the centrosomal accumulation of ciliogenic membrane vesicles. How these distinct processes are properly regulated remains unknown. Here we show that miR-129-3p, a microRNA conserved in vertebrates, controlled cilia biogenesis in cultured cells by concomitantly downregulating CP110 and repressing branched F-actin formation. Blocking miR-129-3p inhibited serum-starvation-induced ciliogenesis, whereas its overexpression potently induced ciliation in proliferating cells and also promoted cilia elongation. Gene expression analysis further identified ARP2, TOCA1, ABLIM1 and ABLIM3 as its targets in ciliation-related actin dynamics. Moreover, miR-129-3p inhibition in zebrafish embryos suppressed ciliation in Kupffer's vesicle and the pronephros, and induced developmental abnormalities including a curved body, pericardial oedema and defective left-right asymmetry. Therefore, our results reveal a mechanism that orchestrates both the centriole-to-basal body transition and subsequent cilia assembly through microRNA-mediated post-transcriptional regulation.

Nudel binds Cdc42GAP to modulate Cdc42 activity at the leading edge of migrating cells.

Cdc42GAP promotes inactivation of Cdc42, a small GTPase whose activation at the leading edge by guanine nucleotide exchange factors is critical for cell migration. How Cdc42GAP is regulated to ensure proper levels of active Cdc42 is poorly understood. Here we show that Nudel, a cytoplasmic dynein regulator, competes with Cdc42 for binding Cdc42GAP. Consequently, Nudel can inhibit Cdc42GAP-mediated inactivation of Cdc42 in a dose-dependent manner. Both Nudel and Cdc42GAP exhibit leading-edge localization in migrating cells. The localization of Nudel requires its phosphorylation by Erk1/2. Depleting Nudel by RNAi or overexpression of a nonphosphorylatable mutant abolishes Cdc42 activation and cell migration. Our data thus uncover Nudel as a regulator of Cdc42 during cell migration. Nudel facilitates cell migration by sequestering Cdc42GAP at the leading edge to stabilize active Cdc42 in response to extracellular stimuli. Excess active Cdc42 may in turn control its own activity by recruiting Cdc42GAP from Nudel.