CasSABER for Programmable In Situ Visualization of Low and Nonrepetitive Gene Loci.

Site-specific imaging of target genes using CRISPR probes is essential for understanding the molecular mechanisms of gene function and engineering tools to modulate its downstream pathways. Herein, we develop CRISPR/Cas9-mediated signal amplification by exchange reaction (CasSABER) for programmable in situ imaging of low and nonrepetitive regions of the target gene in the cell nucleus. The presynthesized primer-exchange reaction (PER) probe is able to hybridize multiple fluorophore-bearing imager strands to specifically light up dCas9/sgRNA target-bound gene loci, enabling in situ imaging of fixed cellular gene loci with high specificity and signal-to-noise ratio. In combination with a multiround branching strategy, we successfully detected nonrepetitive gene regions using a single sgRNA. As an intensity-codable and orthogonal probe system, CasSABER enables the adjustable amplification of local signals in fixed cells, resulting in the simultaneous visualization of multicopy and single-copy gene loci with similar fluorescence intensity. Owing to avoiding the complexity of controlling in situ mutistep enzymatic reactions, CasSABER shows good reliability, sensitivity, and ease of implementation, providing a rapid and cost-effective molecular toolkit for studying multigene interaction in fundamental research and gene diagnosis.

Glycemic Variability and the Risk of Diabetic Peripheral Neuropathy: A Meta-Analysis.

Glycemic variability (GV) has been related to complications in patients with diabetes. The aim of the systematic review and meta-analysis was to investigate whether GV is also associated with the incidence of diabetic peripheral neuropathy (DPN). A systematic search of Medline, Web of Science, Embase, and Cochrane Library database was conducted to identify relevant observational studies with longitudinal follow-up. The Newcastle-Ottawa Scale was used for study quality evaluation. A random-effects model was utilized to pool the results, accounting for heterogeneity. Ten observational studies including 72 565 patients with diabetes were included. The quality score was 8-9, indicating generally good quality of the included studies. With a mean follow-up duration of 7.1 years, 11 532 patients (15.9%) were diagnosed as DPN. Compared to patients with low GV, patients with high GV were associated with an increased risk incidence of DPN (risk ratio: 1.51, 95% confidence interval: 1.23 to 1.85, p<0.001; I2=78%). In addition, subgroup analysis showed consistent results in patients with type 1 and type 2 diabetes, and in studies evaluating the short-term and long-term GV (p for subgroup difference=0.82 and 0.53). Finally, results of subgroup analysis also suggested that the association between GV and risk of DPN were not significantly affected by study design, follow-up durations, diagnostic methods for DPN, adjustment of mean glycated hemoglobin A1c, or study quality scores (p for subgroup difference all>0.05). A high GV may be associated with an increased incidence of DPN.

In Vivo Activation of T-Cell Proliferation by Regulating Cell Surface Receptor Clustering Using a pH-Driven Interlocked DNA Nano-Spring.

Activation of T-cell proliferation specifically in a tumor is crucial for reducing the autoimmune side effects of antitumor immunotherapy. Herein, we developed a pH-driven interlocked DNA nano-spring (iDNS) to stimulate T-cell activation in vivo in response to the low pH value in a tumor microenvironment. The interlocked structure of iDNS provide a more rigid scaffold in comparison to double-stranded DNA for ligand assembly, which can help to control the spatial distribution of ligands with more accuracy. We have demonstrated that the pH-driven reversible reconfiguration of iDNS provides a powerful way to regulate the nanoscale distribution of T-cell receptors (CD3) on the cell surface. The relatively low pH value (pH 6.5) in a solid tumor was able to drive the springlike shrinking of iDNS and induce significant T-cell proliferation, leading to an enhanced antitumor effect, thus providing a tool for specifically inducing an immune response in a tumor for immunotherapy.

Visualizing Single-Nucleotide Variations in a Nuclear Genome Using Colocalization of Dual-Engineered CRISPR Probes.

Direct visualization of single-nucleotide variation (SNV) in single cells is of great importance for understanding the spatial organization of genomes and their relationship with cell phenotypes. Herein, we developed a new strategy for visualizing SNVs in a nuclear genome using colocalization of dual-engineered CRISPR probes (CoDEC). By engineering the structure of sgRNA, we incorporated a hairpin in the spacer domain for improving SNV recognition specificity and a loop in the nonfunctional domain for localized signal amplification. Using guide probe-based colocalization strategy, we can successfully distinguish on-target true positive signals from the off-target false positives with high accuracy. Comparing with a proximity ligation-based assay (CasPLA), the probe colocalization strategy extended applicable target gene sites (the distance between two designed probes can be extended to around 200nt) and improved detection efficiency. This newly developed method provides a facile way for studying in situ information on SNVs in individual cells for basic research and clinical applications with single-molecule and single-nucleotide resolutions.

Discovery of a novel ALK/ROS1/FAK inhibitor, APG-2449, in preclinical non-small cell lung cancer and ovarian cancer models.

BACKGROUND: Tyrosine kinase inhibitors (TKIs) are mainstays of cancer treatment. However, their clinical benefits are often constrained by acquired resistance. To overcome such outcomes, we have rationally engineered APG-2449 as a novel multikinase inhibitor that is highly potent against oncogenic alterations of anaplastic lymphoma kinase (ALK), ROS proto-oncogene 1 receptor tyrosine kinase (ROS1), and focal adhesion kinase (FAK). Here we present the preclinical evaluation of APG-2449, which exhibits antiproliferative activity in cells carrying ALK fusion or secondary mutations. METHODS: KINOMEscan® and LANCE TR-FRET were used to characterize targets and selectivity of APG-2449. Water-soluble tetrazolium salt (WST-8) viability assay and xenograft tumorigenicity were employed to evaluate therapeutic efficacy of monotherapy or drug combination in preclinical models of solid tumors. Western blot, pharmacokinetic, and flow cytometry analyses, as well as RNA sequencing were used to explore pharmacokinetic-pharmacodynamic correlations and the mechanism of actions driving drug combination synergy. RESULTS: In mice bearing wild-type or ALK/ROS1-mutant non-small-cell lung cancer (NSCLC), APG-2449 demonstrates potent antitumor activity, with correlations between pharmacokinetics and pharmacodynamics in vivo. Through FAK inhibition, APG-2449 sensitizes ovarian xenograft tumors to paclitaxel by reducing CD44+ and aldehyde dehydrogenase 1-positive (ALDH1+) cancer stem cell populations, including ovarian tumors insensitive to carboplatin. In epidermal growth factor receptor (EGFR)-mutated NSCLC xenograft models, APG-2449 enhances EGFR TKI-induced tumor growth inhibition, while the ternary combination of APG-2449 with EGFR (osimertinib) and mitogen-activated extracellular signal-regulated kinase (MEK; trametinib) inhibitors overcomes osimertinib resistance. Mechanistically, phosphorylation of ALK, ROS1, and FAK, as well as their downstream components, is effectively inhibited by APG-2449. CONCLUSIONS: Taken together, our studies demonstrate that APG-2449 exerts potent and durable antitumor activity in human NSCLC and ovarian tumor models when administered alone or in combination with other therapies. A phase 1 clinical trial has been initiated to evaluate the safety and preliminary efficacy of APG-2449 in patients with advanced solid tumors, including ALK+ NSCLC refractory to earlier-generation ALK inhibitors. TRIAL REGISTRATION: Clinicaltrial.gov registration: NCT03917043 (date of first registration, 16/04/2019) and Chinese clinical trial registration: CTR20190468 (date of first registration, 09/04/2019).

Downregulation of c-Myc expression confers sensitivity to CHK1 inhibitors in hematologic malignancies.

Checkpoint kinase 1 inhibitors (CHK1i) have shown impressive single-agent efficacy in treatment of certain tumors, as monotherapy or potentiators of chemotherapy in clinical trials, but the sensitive tumor types and downstream effectors to dictate the therapeutic responses to CHK1i remains unclear. In this study we first analyzed GDSC (Genomics of Drug Sensitivity in Cancer) and DepMap database and disclosed that hematologic malignancies (HMs) were relatively sensitive to CHK1i or CHK1 knockdown. This notion was confirmed by examining PY34, a new and potent in-house selective CHK1i, which exhibited potent anti-HM effect in vitro and in vivo, as single agent. We demonstrated that the downregulation of c-Myc and its signaling pathway was the common transcriptomic profiling response of sensitive HM cell lines to PY34, whereas overexpressing c-Myc could partially rescue the anticancer effect of PY34. Strikingly, we revealed the significant correlations between downregulation of c-Myc and cell sensitivity to PY34 in 17 HM cell lines and 39 patient-derived cell (PDC) samples. Thus, our results demonstrate that HMs are more sensitive to CHK1i than solid tumors, and c-Myc downregulation could represent the CHK1i efficacy in HMs.

Copper-based metal-organic framework impedes triple-negative breast cancer metastasis via local estrogen deprivation and platelets blockade.

Metastasis is one of the main causes of failure in the treatment of triple-negative breast cancer (TNBC). Abnormally estrogen level and activated platelets are the key driving forces for TNBC metastasis. Herein, an "ion/gas" bioactive nanogenerator (termed as IGBN), comprising a copper-based MOF and loaded cisplatin-arginine (Pt-Arg) prodrug is developed for metastasis-promoting tumor microenvironment reprogramming and TNBC therapy. The copper-based MOF not only serves as a drug carrier, but also specifically produces Cu2+ in tumors, which catalytic oxidizing estrogen to reduce estrogen levels in situ. Meanwhile, the rationally designed Pt-Arg prodrug reduced into cisplatin to significantly promote the generation of H2O2 in the tumor, then permitting self-augmented cascade NO gas generation by oxidizing Arg through a H2O2 self-supplied way, thus blocking platelet activation in tumor. We clarified that IGBN inhibited TNBC metastasis through local estrogen deprivation and platelets blockade, affording 88.4% inhibition of pulmonary metastasis in a 4T1 mammary adenocarcinoma model. Notably, the locally copper ion interference, NO gas therapy and cisplatin chemotherapy together resulted in an enhanced therapeutic efficacy in primary tumor ablation without significant toxicity. This "ion/gas" bioactive nanogenerator offers a robust and safe strategy for TNBC therapy.

Boosting cisplatin chemotherapy by nanomotor-enhanced tumor penetration and DNA adducts formation.

Despite many nano-based strategies devoted to delivering cisplatin for tumor therapy, its clinical benefits are compromised by poor tissue penetration and limited DNA adducts formation of the drug. Herein, a cisplatin loading nanomotor based janus structured Ag-polymer is developed for cisplatin delivery of deeper tissue and increased DNA adducts formation. The nanomotor displayed a self-propelled tumor penetration fueled by hydrogen peroxide (H2O2) in tumor tissues, which is catalytically decomposed into a large amount of oxygen bubbles by Ag nanoparticles (NPs). Notably, cisplatin could elevate the intracellular H2O2 level through cascade reactions, further promote the degradation of Ag NPs accompanied with the Ag+ release, which could downregulate intracellular Cl- through the formation of AgCl precipitate, thereby enhancing cisplatin dechlorination and Pt-DNA formation. Moreover, polymer can also inhibit the activity of ALKBH2 (a Fe2+-dependent DNA repair enzyme) by chelating intracellular Fe2+ to increase the proportion of irreparable Pt-DNA cross-links. It is found that deep tissue penetration, as well as the increased formation and maintenance of Pt-DNA adducts induced by the nanomotor afford 80% of tumor growth inhibition with negligible toxicity. This work provides an important perspective of resolving chemotherapeutic barriers for boosting cisplatin therapy.

CRISPR/Cas14a-Based Isothermal Amplification for Profiling Plant MicroRNAs.

MicroRNAs (miRNAs) play key roles in biological processes in plants, such as stress resistance, yet can hardly be quantified by an enzyme-involved terminal polymerization process due to their 2'-O-methyl modifications at the 3'-terminal. Herein, we proposed a CRISPR/Cas14a-based rolling circle amplification (termed Cas14R) assay, allowing reverse transcription-free and demethylation-free detection of plant miRNAs with single-nucleotide resolution. The employment of target-templated rolling circle amplification circumvents the extension of the unaccessible 2'-O-methyl group at the 3'-terminal. Particularly, the activated Cas14a confers the trans-cleavage activity for identifying target single-stranded DNA sequences without the necessity of the protospacer adjacent motif, generalizing the detection of miRNA sequences and the integration of different isothermal amplification techniques. Ultimately, the Cas14R assay has been applied to profile miR156a to evaluate the ripeness process of banana, indicating its feasibility in analyzing the roles of miRNAs in biological processes of plants.

Detection of SARS-CoV-2 and Its Mutated Variants via CRISPR-Cas13-Based Transcription Amplification.

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a global health emergency, and its gene mutation and evolution further posed uncertainty of epidemic risk. Herein, we reported a light-up CRISPR-Cas13 transcription amplification method, which enables the detection of SARS-CoV-2 and its mutated variants. Sequence specificity was ensured by both the ligation process and Cas13a/crRNA recognition, allowing us to identify viral RNA mutation. Light-up RNA aptamer allows sensitive output of amplification signals via target-activated ribonuclease activity of CRISPR-Cas13a. The RNA virus assay has been designed to detect coronavirus, SARS-CoV-2, Middle East respiratory syndrome (MERS), and SARS, as well as the influenza viruses such as, H1N1, H7N9, and H9N2. It was accommodated to sense as low as 82 copies of SARS-CoV-2. Particularly, it allowed us to strictly discriminate key mutation of the SARS-CoV-2 variant, D614G, which may induce higher epidemic and pathogenetic risk. The proposed RNA virus assays are promising for point-of-care monitoring of SARS-CoV-2 and its risking variants.

Photoactivated Self-Disassembly of Multifunctional DNA Nanoflower Enables Amplified Autophagy Suppression for Low-Dose Photodynamic Therapy.

Low-dose photodynamic therapy (PDT) holds great promise for reducing undesired patient photosensitivity in cancer treatment. Yet, its therapeutic effect is significantly affected by intracellular cytoprotective processes, such as autophagy. Here, an efficient autophagy suppressor is developed, which is a multifunctional DNA nanoflower (DNF) consisted of tumor-targeting aptamers and DNAzymes for silencing autophagy-related genes, with surface modification of low-dose photosensitizer (Ce6). It is found that the multifunctional DNF can specifically target tumor cells and generate reactive oxygen species (ROS) under light irradiation to trigger self-disassembly of DNF, enhancing the bioavailability of encoded DNAzymes, leading to amplified autophagy suppression. As a facile spatiotemporally programmable photogene therapy platform, the designed DNF is able to suppress tumor growth in vivo with a very low injection dose of Ce6 (18 µg kg-1 , around 100 times lower than the generally applied dose), representing a promising strategy for cancer therapy with safely low-dose PDT.

Lighting up single-nucleotide variation in situ in single cells and tissues.

The ability to 'see' genetic information directly in single cells can provide invaluable insights into complex biological systems. In this review, we discuss recent advances of in situ imaging technologies for visualizing the subtlest sequence alteration, single-nucleotide variation (SNV), at single-cell level. The mechanism of recently developed methods for SNV discrimination are summarized in detail. With recent developments, single-cell SNV imaging methods have opened a new door for studying the heterogenous and stochastic genetic information in individual cells. Furthermore, SNV imaging can be used on morphologically preserved tissue, which can provide information on histological context for gene expression profiling in basic research and genetic diagnosis. Moreover, the ability to visualize SNVs in situ can be further developed into in situ sequencing technology. We expect this review to inspire more research work into in situ SNV imaging technologies for investigating cellular phenotypes and gene regulation at single-nucleotide resolution, and developing new clinical and biomedical applications.

Nanoenabled Intracellular Calcium Bursting for Safe and Efficient Reversal of Drug Resistance in Tumor Cells.

Multidrug resistance (MDR) of a tumor is the main cause of failure of clinical chemotherapy. Herein, we report a simple, yet versatile, tumor-targeting "calcium ion nanogenerator" (TCaNG) to reverse drug resistance by inducing intracellular Ca2+ bursting. Consequently, the TCaNG could induce Ca2+ bursting in acidic lysosomes of tumor cells and then reverse drug resistance according to the following mechanisms: (i) Ca2+ specifically accumulates in mitochondria, suppressing cellular respiration and relieving tumor hypoxia, thus inhibiting P-glycoprotein biosynthesis by downregulating HIF-1α expression. (ii) Ca2+-bursting-induced respiratory depression blocks intracellular ATP production, which further leads to the P-gp incompetence. As a result, the TCaNG could decrease the IC50 of DOX to MCF-7/ADR cells by approximately 30 times and reduce the proliferation of drug-resistant tumors by approximately 13 times without obvious side effects. This simple, safe, and effective "Ca2+ bursting" strategy holds the potential for clinical application in tumor treatment.

Bioinspired Nanosponge for Salvaging Ischemic Stroke via Free Radical Scavenging and Self-Adapted Oxygen Regulating.

Either hypoxia in an acute ischemic stroke before thrombolysis or the oxygen-boost after thrombolysis cause a high level of free radicals, resulting in successive injuries to neurocytes. To treat an ischemic stroke, it is needed to scavenge free radicals, combining sequentially regulating hypoxia and oxygen-boost microenvironment. Here, we report an engineered nanosponge (Mn3O4@nanoerythrocyte-T7, MNET) that could remodel the microenvironment of a stroke by self-adapted oxygen regulating and free radical scavenging. With a long circulation time in blood due to the stealth effect of the erythrocyte and preferential accumulation in the infarct site by the assisting of T7 peptide, MNET exerts a distinct therapeutic effect in two stages of an ischemic stroke: (i) before thrombolysis, rescue neurocyte via rapid free radical scavenging and timely oxygen supply; (ii) after thrombolysis, suppress oxygen-boost via oxygen storage, as well as scavenge free radical to avoid reperfusion injury. MNET holds an attractive potential for ischemic stroke treatment via phased regulation of pathological microenvironment.

Single-Cell Imaging of m6 A Modified RNA Using m6 A-Specific In Situ Hybridization Mediated Proximity Ligation Assay (m6 AISH-PLA).

N6 -methyladenosine (m6 A) modification-the most prevalent mammalian RNA internal modification-plays key regulatory roles in mRNA metabolism. Current approaches for m6 A modified RNA analysis limit at bulk-population level, resulting in a loss of spatiotemporal and cell-to-cell variability information. Here we proposed a m6 A-specific in situ hybridization mediated proximity ligation assay (m6 AISH-PLA) for cellular imaging of m6 A RNA, allowing to identify m6 A modification at specific location in RNAs and image m6 A RNA with single-cell and single-molecule resolution. Using m6 AISH-PLA, we investigated the m6 A level and subcellular location of HSP70 RNA103-m6 A in response to heat shock stress, and found an increased m6 A modified ratio and an increased distribution ratio in cytoplasm under heat shock. m6 AISH-PLA can serve in the study of m6 A RNA in single cells for deciphering epitranscriptomic mechanisms and assisting clinical diagnosis.

Highly Sensitive MicroRNA Detection by Coupling Nicking-Enhanced Rolling Circle Amplification with MoS2 Quantum Dots.

In this work, a label-free and highly sensitive fluorescence assay was constructed for microRNA detection. Nicking-enhanced rolling circle amplification (RCA) induced by G-quadruplex formation is coupled with inner filter effect (IFE)-based quenching effects of MoS2 quantum dots (MoS2 QDs). The padlock probe contains a recognition sequence to target microRNA and an accessible nicking site. The padlock probe is cyclized upon hybridization with target microRNA. Sequentially, amplification initiates a production of a long-concatenated sequence of circular probes. Abundant G-quadruplex sequences are produced via the nicking process and then used as the trigger to initiate the next RCA. In the presence of hemin, numerous hemin/G-quadruplex DNAzymes are formed, which catalyze the oxidation of o-phenylenediamine (OPD) into the colored product 2,3-diaminophenazine, resulting in quenching of the fluorescence of MoS2 QDs. This sensing strategy enables detection of microRNA let-7a with high selectivity and a detection limit of 4.6 fM. The as-prepared sensor was applied for detecting microRNA let-7a in dilute human serum samples and achieved a satisfactory recovery rate, demonstrating its potential in clinic diagnosis of microRNA-associated disease and biochemical research.

Recent Progress and Development in Inorganic Halide Perovskite Quantum Dots for Photoelectrochemical Applications.

Inorganic halide perovskite quantum dots (IHPQDs) have recently emerged as a new class of optoelectronic nanomaterials that can outperform the existing hybrid organometallic halide perovskite (OHP), II-VI and III-V groups semiconductor nanocrystals, mainly due to their relatively high stability, excellent photophysical properties, and promising applications in wide-ranging and diverse fields. In particular, IHPQDs have attracted much recent attention in the field of photoelectrochemistry, with the potential to harness their superb optical and charge transport properties as well as spectacular characteristics of quantum confinement effect for opening up new opportunities in next-generation photoelectrochemical (PEC) systems. Over the past few years, numerous efforts have been made to design and prepare IHPQD-based materials for a wide range of applications in photoelectrochemistry, ranging from photocatalytic degradation, photocatalytic CO2 reduction and PEC sensing, to photovoltaic devices. In this review, the recent advances in the development of IHPQD-based materials are summarized from the standpoint of photoelectrochemistry. The prospects and further developments of IHPQDs in this exciting field are also discussed.

Transcriptomic analysis at the first instar larval stage of nonmolting Bombyx mori mutant (a42).

The life cycle of the holometabolous insect Bombyx mori (Linnaeus) consists of the embryo, larva, pupa, and adult stages with six larval molts. Ecdysone and juvenile hormones play important roles in the growth and development of the silkworms. The a42 silkworm mutant is recessive and homozygous lethal by exhibiting a dark-colored and small body size and fails to molt to second instar. We compared the gene expression of a42 mutants with normal individuals at the first larval molting stage to elucidate the physiological influence of the a42 mutation on the growth and development of silkworms. The transcriptomic sequencing results revealed that 1,411 genes are differentially expressed in a42 mutants, compared with wild-type control silkworms, in which 791 genes are upregulated and 620 genes are downregulated. Gene Ontology/Kyoto Encyclopedia of Genes and Genomes analyses identified differentially expressed genes (DEGs) assigned to biological pathways, such as pentose and glucoronate interconversions, glycerolipid metabolism, folate biosynthesis, amino sugar, and nucleotide sugar metabolism. Two hydroxylases of phenylalanine hydroxylase (BmPAH) and tyrosine hydroxylase (BmTh) are upregulated in a42 mutants. The influence of a42 mutation on these DEGs reveals that melanin metabolism plays an important role during the molting process in silkworms.

Epigenetic regulation of myelination in health and disease.

Myelin is lipid-rich structure that is necessary to avoid leakage of electric signals and to ensure saltatory impulse conduction along axons. Oligodendrocytes in central nervous system (CNS) and Schwann cells in peripheral nervous system (PNS) are responsible for myelin formation. Axonal demyelination after injury or diseases greatly impairs normal nervous system function. Therefore, understanding how the myelination process is programmed, coordinated, and maintained is crucial for developing therapeutic strategies for remyelination in the nervous system. Epigenetic mechanisms have been recognized as a fundamental contributor in this process. In recent years, histone modification, DNA modification, ATP-dependent chromatin remodeling, and non-coding RNA modulation are very active area of investigation. We will present a conceptual framework that integrates crucial epigenetic mechanisms with the regulation of oligodendrocyte and Schwann cell lineage progression during development and myelin degeneration in pathological conditions. It is anticipated that a refined understanding of the molecular basis of myelination will aid in the development of treatment strategies for debilitating disorders that involve demyelination, such as multiple sclerosis in the CNS and neuropathies in the PNS.

RNA Strand Displacement Responsive CRISPR/Cas9 System for mRNA Sensing.

CRISPR/Cas9 has already become a powerful tool for genomic manipulation, and further engineering of the system allows it to be precisely regulated in response to external signals, thus, broadening its application possibilities, such as biosensing or bioimaging. However, most stimuli-responsive CRISPR systems are built based on elaborately designed and engineered inducible Cas9 proteins, and external stimuli are still mostly limited as small molecules and light. To construct more precise and easy-to-build responsive CRISPR systems and broaden their responsive species, we seek to engineer conditional guide RNA, rather than Cas9 protein, to mediate conditional CRISPR corresponding to logic operation. Here, we construct mRNA-sensing CRISPR by gRNA reconfiguration and toehold mediated strand displacement, in which each target site could be independently controlled. We show that switches can be embedded into the gRNA and used as RNA sensors, capable of detecting multiple mRNA inputs orthogonally and providing CRISPR/Cas9 response outputs. NOR and NAND logical gates are also constructed, demonstrating its orthogonality and programmability. This strategy promises potential uses in constructing genetic circuits to detect endogenous mRNAs and initiate cellular responses.