Identification of low-abundance proteins in the royal jelly using the Osborne classification method.

Royal jelly (RJ) is recognized as healthy food, with a high content of proteins. These proteins play important roles in honeybee caste and human health, but the proteomic analysis of low-abundance proteins in RJ has long been a challenge. Herein, we used the Osborne classification method to separate the RJ proteins of Xinjiang black bees into various fractions. The globulin, ethanol-soluble protein, and glutelin fractions were further separated by SDS-PAGE, and proteomic analysis was carried out by LC-MS/MS and searched against the UniProt database. A total of 23 secretory proteins were identified by proteomic analysis, in which 7 proteins were identified for the first time in RJ. The Osborne classification method combining one-dimensional gel electrophoresis-based proteomic analysis allows the identification of low-abundance proteins in the RJ and greatly extends the knowledge about the components and functions of RJ proteins. The raw data are available via ProteomeXchange with the identifier PXD023315. SIGNIFICANCE: This study makes an important contribution to the research of the components and functions of low-abundance royal jelly proteins for the following reasons.

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.

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.

DNA nanodevice as a multi-module co-delivery platform for combination cancer immunotherapy.

A major challenge in combining cancer immunotherapy is the efficient delivery of multiple types of immunological stimulators to elicit a robust anti-tumor immune response and reprogram the immunosuppressive tumor microenvironment (TME). Here, we developed a DNA nanodevice that was generated by precisely assembling three types of immunological stimulators. The doxorubicin (Dox) component induced immunogenic cell death (ICD) in tumor cells and enhanced phagocytosis of antigen-presenting cells (APCs). Exogenous double-stranded DNA (dsDNA) could act as a molecular adjuvant to activate the stimulator of interferon genes (STING) signaling in APCs by engulfing dying tumor cells. Interleukin (IL)-12 and small hairpin programmed cell death-ligand 1 (shPD-L1) transcription templates were designed to regulate TME. Additionally, for targeted drug delivery, multiple cyclo[Arg-Gly-Asp-(d-Phe)-Cys] (cRGD) peptide units on DNA origami were employed. The incorporation of disulfide bonds allowed the release of multiple modules in response to intracellular glutathione (GSH) in tumors. The nanodevice promoted the infiltration of CD8+ and CD4+ cells into the tumor and generated a highly inflamed TME, thereby enhancing the effectiveness of cancer immunotherapy. Our research results indicate that the nanodevice we constructed can effectively inhibit tumor growth and prevent lung metastasis without obvious systemic toxicity, providing a promising strategy for cancer combination treatment.

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.

Surgical tumor-derived nanoplatform targets tumor-associated macrophage for personalized postsurgical cancer immunotherapy.

Directly activating CD8+ T cells within the tumor through antigen-presenting cells (APCs) hold promise for tumor elimination. However, M2-like tumor-associated macrophages (TAMs), the most abundant APCs in tumors, hinder CD8+ T cell activation due to inefficient antigen cross-presentation. Here, we demonstrated a personalized nanotherapeutic platform using surgical tumor-derived galactose ligand-modified cancer cell membrane (CM)-coated cysteine protease inhibitor (E64)-loaded mesoporous silica nanoparticles for postsurgical cancer immunotherapy. The platform targeted M2-like TAMs and released E64 within lysosomes, which reshaped antigen cross-presentation and directly activated CD8+ T cells, thus suppressing B16-OVA melanoma growth. Furthermore, this platform, in combination with anti-PD-L1 antibodies, enhanced the therapeutic efficacy and substantially inhibited 4T1 tumor growth. CMs obtained from surgically resected tumors were used to construct a personalized nanotherapeutic platform, which, in synergy with immune checkpoint blockade (ICB), effectively inhibited postsurgical tumor recurrence in 4T1 tumor. Our work offered a robust, safe strategy for cancer immunotherapy and prevention of postsurgical tumor recurrence.

Xanthene-based near-infrared chromophores for high-contrast fluorescence and photoacoustic imaging of dipeptidyl peptidase 4.

Near-infrared (NIR) chromophores with analyte tunable emission and absorption properties are highly desirable for developing activatable fluorescence and photoacoustic (PA) probes for bioimaging and disease diagnosis. Here we engineer a class of new chromophores by extending the π-conjugation system of a xanthene scaffold at position 7 with different electron withdrawing groups. It is demonstrated that these chromophores exhibit pH-dependent transition from a spirocyclic "closed" form to a xanthene "open" form with remarkable changes in spectral properties. We further develop fluorescence and PA probes by caging the NIR xanthene chromophores with a dipeptidyl peptidase 4 (DPPIV) substrate. In vitro and live cell studies show that these probes allow activatable fluorescence and PA detection and imaging of DPPIV activity with high sensitivity, high specificity and fast response. Moreover, these two probes allow high-contrast and highly specific imaging of DPPIV activity in a tumour-bearing mouse model in vivo via systemic administration. This study highlights the potential of a xanthene scaffold as a versatile platform for developing high-contrast fluorescence and PA molecular probes.

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.

Near-infrared fluorogenic imaging of carbon monoxide in live cells using palladium-mediated carbonylation.

Here we develop a near infrared (NIR) fluorogenic probe for carbon monoxide (CO) detection and imaging based on palladium-mediated carbonylation using a NIR boron-dipyrromethene difluoride as a fluorophore and tetraethylene glycols as aqueous moieties. The probe is utilized to image exogenous and endogenous CO under different stimulated conditions in live cells.

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.

In situ monitoring of ROS secretion from single cells with a dual-nanopore biosensor.

We report here a dual-nanopore biosensor based on modulation of surface charge density coupled with a microwell array chip for in situ monitoring of ROS secretion from single MCF-7 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.

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.

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.

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.

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.

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.

Enzyme Regulating the Wettability of the Outer Surface of Nanochannels.

Functional probes not only at the inner wall but also at the outer surface of nanochannel systems could be used for the recognition and detection of biotargets. Despite the advancements, the current detection mechanisms are still mainly based on the surface charge variation. We proposed a strategy of using the variation of wettability on the outer surface of nanochannels for detecting a tumor marker, herein, exemplifying matrix metalloproteinase-2 (MMP-2). The outer surface of the nanochannels were modified with amphipathic peptide probe consisting of hydrophilic unit (CRRRR), MMP-2 cleavage unit (PLGLAG), and hydrophobic unit (Fn). After recognition of MMP-2, due to the release of hydrophobic unit, the hydrophilicity of the outer surface was expected to increase, thus leading to the increase of ion current. Furthermore, the number (n) of phenylalanine (F) in the hydrophobic unit was modulated from 2, 4, to 6. By lengthening the hydrophobic unit, the limit of detection for MMP-2 detection could reach 1 ng/mL (when n = 6) and improve by 50-fold (to n = 2). This nanochannel system was utilized to successfully detect the MMP-2 secreted from cells and demonstrated that the expression of MMP-2 was related to the cell cycle and exhibited the highest level in G1/S phase. This study proved that in addition to the surface charge, wettability regulation could also be utilized as a variation factor to broaden the design strategy of a probe on OS to achieve the detection of biotargets.

Chinese genetic variation database of inborn errors of metabolism: a systematic review of published variants in 13 genes.

BACKGROUND: Population-specific variation database of inborn errors of metabolism (IEMs) is essential for precise genetic diagnosis and disease prevention. Here we presented a systematic review of clinically relevant variants of 13 IEMs genes reported among Chinese patients. METHODS: A systematic search of the following electronic databases for 13 IEMs genes was conducted: PubMed-NCBI, China national knowledge infrastructure and Wanfang databases. Patient data was extracted from articles eligible for inclusion and recorded in Excel electronic form using a case-by-case approach. RESULTS: A total of 218 articles, 93 published in English and 125 in Chinese, were retrieved. After variant annotation and deduplication, 575 unique patients (241 from articles published in Chinese) were included in the population-specific variation database. Patients identified by newborn screening and symptomatic presentation were 231 (40.17%) and 344 (59.83%), respectively. Biallelic variants were observed in 525/575 (91.3%). Among the 581 unique variants identified, 83 (14.28%) were described ≥ 3 times and 97 (16.69%) were not recorded in Clinvar or HGMD. Four variants were reclassified as benign and dozens of confusing variants deserved further research. CONCLUSION: This review provides a unique resource of the well-characterized diseases and causative variants that have accumulated in Chinese population and is a preliminary attempt to build the Chinese genetic variation database of IEMs.