Sources of particulate-matter air pollution and its oxidative potential in Europe.

Particulate matter is a component of ambient air pollution that has been linked to millions of annual premature deaths globally1-3. Assessments of the chronic and acute effects of particulate matter on human health tend to be based on mass concentration, with particle size and composition also thought to play a part4. Oxidative potential has been suggested to be one of the many possible drivers of the acute health effects of particulate matter, but the link remains uncertain5-8. Studies investigating the particulate-matter components that manifest an oxidative activity have yielded conflicting results7. In consequence, there is still much to be learned about the sources of particulate matter that may control the oxidative potential concentration7. Here we use field observations and air-quality modelling to quantify the major primary and secondary sources of particulate matter and of oxidative potential in Europe. We find that secondary inorganic components, crustal material and secondary biogenic organic aerosols control the mass concentration of particulate matter. By contrast, oxidative potential concentration is associated mostly with anthropogenic sources, in particular with fine-mode secondary organic aerosols largely from residential biomass burning and coarse-mode metals from vehicular non-exhaust emissions. Our results suggest that mitigation strategies aimed at reducing the mass concentrations of particulate matter alone may not reduce the oxidative potential concentration. If the oxidative potential can be linked to major health impacts, it may be more effective to control specific sources of particulate matter rather than overall particulate mass.

G-Quadruplex mRNAs Silencing with Inducible Ribonuclease Targeting Chimera for Precision Tumor Therapy.

Ribonuclease targeting chimera (RIBOTAC) represents an emerging strategy for targeted therapy. However, RIBOTAC that is selectively activated by bio-orthogonal or cell-specific triggers has not been explored. We developed a strategy of inducible RIBOTAC (iRIBOTAC) that enables on-demand degradation of G-quadruplex (G4) RNAs for precision cancer therapy. iRIBOTAC is designed by coupling an RNA G4 binder with a caged ribonuclease recruiter, which can be decaged by a bio-orthogonal reaction, tumor-specific enzyme, or metabolite. A bivalent G4 binder is engineered by conjugating a near-infrared (NIR) fluorescence G4 ligand to a noncompetitive G4 ligand, conferring fluorescence activation on binding G4s with synergistically enhanced affinity. iRIBOTAC is demonstrated to greatly knockdown G4 RNAs upon activation under bio-orthogonal or cell-specific stimulus, with dysregulation of gene expressions involving cell killing, channel regulator activity, and metabolism as revealed by RNA sequencing. This strategy also shows a crucial effect on cell fate with remarkable biochemical hallmarks of apoptosis. Mice model studies demonstrate that iRIBOTAC allows selective imaging and growth suppression of tumors with bio-orthogonal and tumor-specific controls, highlighting G4 RNA targeting and inducible silencing as a valuable RIBOTAC paradigm for cancer therapy.

Chemically Inducible DNAzyme Sensor for Controllable Imaging of Metal Ions.

RNA-cleaving DNAzymes have emerged as a promising tool for metal ion detection. Achieving spatiotemporal control over their catalytic activity is essential for understanding the role of metal ions in various biological processes. While photochemical and endogenous stimuli-responsive approaches have shown potential for controlled metal ion imaging using DNAzymes, limitations such as photocytotoxicity, poor tissue penetration, or off-target activation have hindered their application for safe and precise detection of metal ions in vivo. We herein report a chemically inducible DNAzyme in which the catalytic core is modified to contain chemical caging groups at the selected backbone sites through systematic screening. This inducible DNAzyme exhibits minimal leakage of catalytic activity and can be reactivated by small molecule selenocysteines, which effectively remove the caging groups and restore the activity of DNAzyme. Benefiting from these findings, we designed a fluorogenic chemically inducible DNAzyme sensor for controlled imaging of metal ions with tunable activity and high selectivity in live cells and in vivo. This chemically inducible DNAzyme design expands the toolbox for controlling DNAzyme activity and can be easily adapted to detect other metal ions in vivo by changing the DNAzyme module, offering opportunities for precise biomedical diagnosis.

Screening results and mutation frequency analysis of G6PD deficiency in 1,291,274 newborns in Huizhou, China: a twenty-year experience.

OBJECTIVES: This study aimed to investigate the incidence rate and spectrum of gene mutations of Glucose-6-phosphate dehydrogenase (G6PD) deficiency in the Huizhou city of southern China to provide a scientific basis for disease prevention and control in the area. METHODS: From March 2003 to December 2022, newborn screening for G6PD enzyme activity was carried out in Huizhou city using the fluorescence quantitative method. Infants who tested positive during the initial screening were diagnosed using the nitroblue tetrazolium ratio method, while a subset of infants received further gene mutation analysis using the multicolor probe melting curve analysis method. RESULTS: A total of 1,291,274 newborns were screened and the screening rate has increased from 20.39% to almost 100%. In the 20-year period, 57,217 (4.43%) infants testing positive during the initial screening. Out of these infants, 49,779 (87%) were recalled for confirmatory testing. G6PD deficiency was confirmed in 39,261 of the recalled infants, indicating a positive predictive value of 78.87%. The estimated incidence rate of G6PD deficiency in the region was 3.49%, which was significantly higher than the average incidence rate of 2.1% in southern China. On the other hand, seven pathogenic G6PD variants were identified in the analysis of the 99 diagnosed infants with the most common being c.1388 G > A (48.5%), followed by c.95 A > G (19.2%), c.1376 G > T (15.2%), c.871 G > A (9.1%), c.1360 C > T (3.0%), c.392 G > T (3.0%), and c.487 G > A (1.0%). CONCLUSION: The incidence of G6PD deficiency in newborns in the Huizhou city was higher than the southern China average level, while the types and frequencies of gene mutations were found to vary slightly from other regions. Our findings suggested that free government screening and nearby diagnosis strategies could reduce the incidence of G6PD deficiency in the area.

Ligand-Induced Divergent Evolution of ZnSe Magic Sized Clusters.

Alkylamine ligand-induced evolutions of ZnSe magic sized clusters (MSCs) toward divergent products have been discovered for the first time. With correspondingly assigned molecular structures, the same ZnSe MSC was found to undergo either single-atom growth or dissolution through the elaborate tailoring of alkylamine ligands.

Result of a Pilot External Quality Assessment Scheme for Clinical Diagnosis of Inherited Metabolic Disorders in China.

BACKGROUND: We aimed to evaluate the diagnostic capabilities of Chinese laboratories for inherited metabolic disorders (IMDs) using gas chromatography-mass spectrometry (GC-MS) on urine samples. Meanwhile, based on the result of the pilot external quality assessment (EQA) scheme, we hope to establish a standardized and reliable procedure for future EQA practice. METHODS: We recruited laboratories that participated in the EQA of quantitative analysis of urinary organic acids with GC-MS before joining the surveys. In each survey, a set of five real urine samples was distributed to each participant. The participants should analyze the sample by GC-MS and report the "analytical result", "the most likely diagnosis", and "recommendation for further tests" to the NCCL before the deadline. RESULTS: A total of 21 laboratories participated in the scheme. The pass rates were 94.4% in 2020 and 89.5% in 2021. For all eight IMDs tested, the analytical proficiency rates ranged from 84.7% - 100%, and the interpretational performance rate ranged from 88.2% - 97.0%. The performance on hyperphenylalaninemia (HPA), 3-methylcrotonyl-CoA carboxylase deficiency (MCCD), and ethylmalonic encephalopathy (EE) samples were not satisfactory. CONCLUSIONS: In general, the participants of this pilot EQA scheme are equipped with the basic capability for qualitative organic acid analysis and interpretation of the results. Limited by the small size of laboratories and samples involved, this activity could not fully reflect the state of clinical practice of Chinese laboratories. NCCL will improve the EQA scheme and implement more EQA activities in the future.

Results of a Pilot External Quality Assessment Scheme for Genetic Testing of Newborns with Spinal Muscular Atrophy.

BACKGROUND: This study aims to evaluate the ability of laboratories to perform spinal muscular atrophy (SMA) genetic testing in newborns based on dried blood spot (DBS) samples, and to provide reference data and advance preparation for establishing the pilot external quality assessment (EQA) scheme for SMA genetic testing of newborns in China. METHODS: The pilot EQA scheme contents and evaluation principles of this project were designed by National Center for Clinical Laboratories (NCCL), National Health Commission. Two surveys were carried out in 2022, and 5 batches of blood spots were submitted to the participating laboratory each time. All participating laboratories conducted testing upon receiving samples, and test results were submitted to NCCL within the specified date. RESULTS: The return rates were 75.0% (21/28) and 95.2% (20/21) in the first and second surveys, respectively. The total return rate of the two examinations was 83.7% (41/49). Nineteen laboratories (19/21, 90.5%) had a full score passing on the first survey, while in the second survey twenty laboratories (20/20, 100%) scored full. CONCLUSIONS: This pilot EQA survey provides a preliminary understanding of the capability of SMA genetic testing for newborns across laboratories in China. A few laboratories had technical or operational problems in testing. It is, therefore, of importance to strengthen laboratory management and to improve testing capacity for the establishment of a national EQA scheme for newborn SMA genetic testing.

Targeted Protein Degradation Mediated by Genetically Engineered Lysosome-Targeting Exosomes.

Protein-degrading chimeras are superior drug modalities compared to traditional protein inhibitors because of their effective therapeutic performance. So far, various targeted protein degradation strategies, including proteolysis-targeting chimeras and lysosome-targeting chimeras, have emerged as essential technologies for tackling diseases caused by abnormal protein expression. Here, we report the development and application of lysosome-targeting exosomes (LYTEXs) for the selective degradation of membrane protein targets. LYTEXs are genetically engineered exosomes expressing multivalent single-chain fragment variables, simultaneously recognizing cell-surface lysosome-targeting and to-be-degraded protein. We show that by targeting the lysosome-directing asialoglycoprotein receptor, bispecific LYTEXs can induce lysosomal degradation of membrane-associated therapeutic targets. This strategy provides a generalizable, easy-to-prepare platform for modulating surface protein expression, with the advantage of therapeutic delivery.

Site-Specific Bioorthogonal Activation of DNAzymes for On-Demand Gene Therapy.

RNA-cleaving DNAzymes hold great promise as gene silencers, and spatiotemporal control of their activity through site-specific reactions is crucial but challenging for on-demand therapy. We herein report a novel design of a bioorthogonally inducible DNAzyme that is deactivated by site-specific installation of bioorthogonal caging groups on the designated backbone sites but restores the activity via a phosphine-triggered Staudinger reduction. We perform a systematical screening for installing the caging groups on each backbone site in the catalytic core of 10-23 DNAzyme and identify an inducible DNAzyme with very low leakage activity. This design is demonstrated to achieve bioorthogonally controlled cleavage of exogenous and endogenous mRNA in live cells. It is further extended to photoactivation and endogenous stimuli activation for spatiotemporal or targeted control of gene silencing. The bioorthogonally inducible DNAzyme is applied to a triple-negative breast cancer mouse model using a lipid nanoparticle delivery system, demonstrating high efficiency in knockdown of Lcn2 oncogenes and substantial suppression of tumor growth, thus highlighting the potential of precisely controlling the DNAzyme functions for on-demand gene therapy.

Genetically Encoded RNA Sensors for Ratiometric and Multiplexed Imaging of Small Molecules in Living Cells.

Genetically encoded sensors afford powerful tools for studying small molecules and metabolites in live cells. However, genetically encoded sensors with a general design remain to be developed. Here we develop genetically encoded RNA sensors with a modular design for ratiometric and multiplexed imaging of small molecules in live cells. The sensor utilizes aptazyme as a recognition module and the light-up RNA aptamer as a signal reporter. The conformation of light-up aptamers is abrogated by a blocking sequence, and aptazyme-mediated cleavage restores the correct conformation, delivering activated fluorescence for small molecule imaging. We first developed a genetically encoded ratiometric sensor using Mango aptamer as a reference and SRB2 as a reporter. It is shown that the sensor allows quantitative imaging and detection of theophylline in live cells. The generality of the design is further demonstrated for imaging other small molecules by replacing the aptazymes. Its ability for multiplexed imaging of small molecules is further explored via the integration of different small-molecule responsive aptazymes and light-up RNA aptamers. This modular design could offer a versatile platform for imaging diverse molecules in living cells.

In Situ Transformable Pro-nanotheranostic Platform for Activable Photoacoustic Imaging and Synergistic Photothermal/Chemodynamic Cancer Therapy.

Nanotheranostic platforms integrated with diagnostic and therapeutic functions have been widely developed for tumor medicine. However, the "always-on" nanotheranostic platforms suffer from poor tumor specificity, which may largely restrict therapeutic efficacy and prevent precise theranostics. Here, we develop an in situ transformable pro-nanotheranostic platform (ZnS/Cu2O@ZIF-8@PVP) by encapsulating ZnS and Cu2O nanoparticles in a metal-organic framework (MOF) nanomaterial of ZIF-8 that allows activable photoacoustic (PA) imaging and synergistic photothermal/chemodynamic therapy (PTT/CDT) of tumors in vivo. It is shown that the pro-nanotheranostic platform gradually decomposes and releases ZnS nanoparticles and Cu+ ions in acidic conditions, which spontaneously trigger a cation exchange reaction and synthesize Cu2S nanodots in situ with activated PA signals and PTT effects. Moreover, the excessive Cu+ ions function as Fenton-like catalysts and catalyze the production of highly reactive hydroxyl radicals (•OH) for CDT using elevated levels of H2O2 in tumor microenvironments (TMEs). In vivo studies demonstrate that the in situ transformable pro-nanotheranostic platform can specifically image tumors via PA and photothermal imaging and efficiently ablate tumors through synergistic CDT/PTT. Our in situ transformable pro-nanotheranostic platform could provide a new arsenal for precise theranostics in cancer therapy.

Reversible Acylation of RNA Enables Activatable Biosensing.

There is a high demand to develop chemical tools to control the property and function of RNA. Current methods mainly rely on ultraviolet light-based caging strategies, which may cause phototoxicity in live cell-based experiments. We herein report an endogenous stimulus-responsive RNA acylation approach by introducing boronate ester (BE) groups to 2'-hydroxyls through postsynthetic modification. Treatment with hydrogen peroxide (H2O2) yields a phenol derivative which undergoes a 1,6-eliminaton for the traceless release of 2'-hydroxyl. We demonstrated that the acylation of crRNA enabled conditional regulation of CRISPR/Cas13a activity for activatable detection of target RNA. We also showed that the highly specific acylation of the single RNA in 8-17 DNAzyme allowed reversible control of the catalytic activity of DNAzyme, which was further applied to the cell-selective imaging of metal ions in cancer cells. Thus, our strategy provides a simple, general, and cell-selective method to control RNA activity, affording great potential in the construction of activatable RNA sensors and pre-RNA medicines.

Protein-Scaffolded DNA Nanostructures for Imaging of Apurinic/Apyrimidinic Endonuclease 1 Activity in Live Cells.

Nucleic acids are valuable tools for intracellular biomarker detection and gene regulation. Here we propose a new type of protein (avidin)-scaffolded DNA nanostructure (ADN) for imaging the activity of apurinic/apyrimidinic endonuclease 1 (APE1) in live cells. ADN is designed by assembling an avidin-displayed abasic site containing DNA strands labeled with a fluorophore or a quencher via a complementary linker strand. ADN is nonemissive due to the close proximity of fluorophores and quenchers. APE1-mediated cleavage separates the fluorophores from the quenchers, delivering activated fluorescence. In vitro assays show that ADN is responsive to APE1 with high sensitivity and high specificity. ADN can efficiently enter the cells, and its capability to visualize and detect intracellular APE1 activities is demonstrated in drug-treated cells and different cell lines. The modular and easy preparation of our nanostructures would afford a valuable platform for imaging and detecting APE1 activities in live cells.

Cell-Specific Degradation of Histone Deacetylase Using Warhead-Caged Proteolysis Targeting Chimeras.

Proteolysis targeting chimeras (PROTACs) have shifted the paradigm for drug development via target protein degradation. However, PROTACs may exhibit systemic toxicity to normal cells due to indiscriminate degradation and the utility of inhibitors as a warhead for protein targeting. Here, we propose a new strategy for developing activatable PROTACs for cell-specific degradation of histone deacetylase (HDAC) with minimal side effects via caging of the warhead. Molecular docking reveals that the hydroxyl group of the HDAC inhibitor is crucial for targeting. An enzyme-activatable PROTAC is designed by caging the hydroxyl group with the substrate for NAD(P)H: quinone oxidoreductase 1 (NQO1) overexpressed in cancer cells. We demonstrate that the caged PROTAC can be converted to its active form in response to NQO1. The enzyme-activatable PROTAC allows the efficient and specific degradation of HDAC6 and exerts antiproliferative activity in NQO1-positive cells. The generalizability of the design is further demonstrated by engineering a H2O2-responsive PROTAC for specific degradation of HDAC6 in cells with elevated H2O2. The strategy of caging the ligand for target proteins would afford a new dimension for developing activatable PROTACs with high specificity and minimal side effects.

Selective Proteolysis of Activated Transcriptional Factor by NIR-Responsive Palindromic DNA Thalidomide Conjugate Inhibits the Canonical Smad Pathway.

Dysfunctional transcription factors that activate abnormal expressions of specific proteins are often associated with the progression of various diseases. Despite being attractive drug targets, the lack of druggable sites has dramatically hindered their drug development. The emergence of proteolysis targeting chimeras (PROTACs) has revitalized the drug development of many conventional hard-to-drug protein targets. Here, the use of a palindromic double-strand DNA thalidomide conjugate (PASTE) to selectively bind and induce proteolysis of targeted activated transcription factor (PROTAF) is reported. The selective proteolysis of the dimerized phosphorylated receptor-regulated Smad2/3 and inhibition of the canonical Smad pathway validates PASTE-mediated PROTAF. Further aptamer-guided active delivery of PASTE and near-infrared light-triggered PROTAF are demonstrated. Great potential in using PASTE for the selective degradation of the activated transcription factor is seen, providing a powerful tool for studying signaling pathways and developing precision medicines.

Type I Photosensitizer Targeting G-Quadruplex RNA Elicits Augmented Immunity for Cancer Ablation.

Type I photodynamic therapy (PDT) represents a promising treatment modality for tumors with intrinsic hypoxia. However, type I photosensitizers (PSs), especially ones with near infrared (NIR) absorption, are limited and their efficacy needs improvement via new targeting tactics. We develop a NIR type I PS by engineering acridinium derived donor-π-acceptor systems. The PS exhibits an exclusive type I PDT mechanism due to effective intersystem crossing and disfavored energy transfer to O2 , and shows selective binding to G-quadruplexes (G4s) via hydrogen bonds identified by a molecular docking study. Moreover, it enables fluorogenic detection of G4s and efficient O2 ⋅- production in hypoxic conditions, leading to immunogenic cell death and substantial variations of gene expression in RNA sequencing. Our strategy demonstrates augmented antitumor immunity for effective ablation of immunogenic cold tumor, highlighting its potential of RNA-targeted type I PDT in precision cancer therapy.

Ligand Dilution Analysis Facilitates Aptamer Binding Characterization at the Single-Molecule Level.

Cell-specific aptamers offer a powerful tool to study membrane receptors at the single-molecule level. Most target receptors of aptamers are highly expressed on the cell surface, but difficult to analyze in situ because of dense distribution and fast velocity. Therefore, we herein propose a random sampling-based analysis strategy termed ligand dilution analysis (LDA) for easily implemented aptamer-based receptor study. Receptor density on the cell surface can be calculated based on a regression model. By using a synergistic ligand dilution design, colocalization and differentiation of aptamer and monoclonal antibody (mAb) binding on a single receptor can be realized. Once this is accomplished, precise binding site and detailed aptamer-receptor binding mode can be further determined using molecular docking and molecular dynamics simulation. The ligand dilution strategy also sets the stage for an aptamer-based dynamics analysis of two- and three-dimensional motion and fluctuation of highly expressed receptors on the live cell membrane.

A DNA Logic Circuit Equipped with a Biological Amplifier Loaded into Biomimetic ZIF-8 Nanoparticles Enables Accurate Identification of Specific Cancers In Vivo.

DNA logic circuits (DLC) enable the accurate identification of specific cell types, such as cancer cells, but they face the challenges of weak output signals and a lack of competent platforms that can efficiently deliver DLC components to the target site in the living body. To address these issues, we rationally introduced a cascaded biological amplifier module based on the Primer Exchange Reaction inspired by electronic circuit amplifier devices. As a paradigm, three abnormally expressed Hela cell microRNAs (-30a, -17, and -21) were chosen as "AND" gate inputs. DLC response to these inputs was boosted by the amplifier markedly enhancing the output signal. More importantly, the encapsulation of DLC and amplifier components into ZIF-8 nanoparticles resulted in their efficient delivery to the target site, successfully distinguishing the Hela tumor subtype from other tumors in vivo. Thus, we envision that this strategy has great potential for clinical cancer diagnosis.

DNA-Templated Bioorthogonal Reactions via Catalytic Hairpin Assembly for Precise RNA Imaging in Live Cells.

There has been a significant interest in developing proximity-induced bioorthogonal reactions for nucleic acid detection and imaging, owing to their high specificity and tunable reaction kinetics. Herein, we reported the first design of a fluorogenic sensor by coupling a bioorthogonal reaction with a DNA cascade circuit for precise RNA imaging in live cells. Two DNA hairpin probes bearing tetrazines or vinyl ether caged fluorophores were designed and synthesized. Upon target mRNA triggering catalytic hairpin assembly, the chemical reaction partners were brought in a spatial proximity to yield high effective concentrations, which dramatically facilitated the bioorthogonal reaction efficiency to unmask the vinyl ether group to activate fluorescence. The proposed fluorogenic sensor was demonstrated to have a high signal-to-noise ratio up to ∼30 fold and enabled the sensitive detection of target mRNA with a detection limit of 4.6 pM. Importantly, the fluorogenic sensor presented low background signals in biological environments due to the unique "click to release" feature, avoiding false positive results caused by unspecific degradation. We also showed that the fluorogenic sensor could accurately image mRNA in live cells and distinguish the relative mRNA expression levels in both tumor and normal cells. Benefiting from these significant advantages, our method provides a useful tool for basic studies of bioorthogonal chemistry and early clinical diagnosis.

Single Molecule-Level Detection via Liposome-Based Signal Amplification Mass Spectrometry Counting Assay.

Because of the low atomization and/or ionization efficiencies of many biological macromolecules, the application of mass spectrometry to the direct quantitative detection of low-abundance proteins and nucleic acids remains a significant challenge. Herein, we report mass spectrum tags (MS-tags) based upon gold nanoparticle (AuNP)-templated phosphatidylcholine phospholipid (DSPC) liposomes, which exhibit high and reliable signals via electrospray ionization (ESI). Using these MS-tags, we constructed a liposome signal amplification-based mass spectrometric (LSAMS) "digital" counting assay to enable ultrasensitive detection of target nucleic acids. The LSAMS system consists of liposomes modified with a gold nanoparticle core and surface-anchored photocleavable DNA. In the presence of target nucleic acids, the modified liposome and a magnetic bead simultaneously hybridize with the target nucleic acid. After magnetic separation and photolysis, the MS-tag is released and can be analyzed by ESI-MS. At very low target concentrations, one liposome particle corresponds to one target molecule; thus, the concentration of the target can be estimated by counting the number of liposomes. With this assay, hepatitis C (HCV) virus RNA was successfully analyzed in clinical samples.