A DNA walker based on hairpin-shaped DNA aligner and fueled by nicking endonuclease for sensitive and rapid miRNA analysis.

BACKGROUND: DNA walker-based strategies have gained significant attention in nucleic acid analysis. However, they face challenges related to balancing design complexity, sequence dependence, and amplification efficiency. Furthermore, most existing DNA walkers rely on walking and lock probes, requiring optimization of various parameters like DNA probe sequence, walking-to-lock probe ratio, lock probe length, etc. to achieve optimal performance. This optimization process is time-consuming and adds complexity to experiments. To enhance the performance and reliability of DNA walker nanomachines, there is a need for a simpler, highly sensitive, and selective alternative strategy. RESULTS: A sensitive and rapid miRNA analysis strategy named hairpin-shaped DNA aligner and nicking endonuclease-fueled DNA walker (HDA-NE DNA walker) was developed. The HDA-NE DNA walker was constructed by modifying hairpin-shaped DNA aligner (HDA) probe and substrate report (SR) probe on the surface of AuNPs. Under normal conditions, HDA and SR remained stable. However, in the presence of miR-373, HDA underwent a conformational transition to an activated structure to continuously cleave the SR probe on the AuNPs with the assistance of Nt.AlwI nicking endonuclease, resulting in sensitive miRNA detection with a detection limit as low as 0.23 pM. Additionally, the proposed HDA-NE DNA walker exhibited high selectivity in distinguishing miRNAs with single base differences and can effectively analyze miR-373 levels in both normal and breast cancer patient serums. SIGNIFICANCE: The proposed HDA-NE DNA walker system was activated by a conformational change of HDA probe only in the presence of the target miRNA, eliminating the need for a lock probe and without sequence dependence for SR probe. This strategy demonstrated a rapid reaction rate of only 30 min, minimal background noise, and a high signal-to-noise ratio (S/B) compared to capture/lock-based DNA walker. The method is expected to become a powerful tool and play an important role in disease diagnosis and precision therapy.

CRISPR/Pepper-tDeg: A Live Imaging System Enables Non-Repetitive Genomic Locus Analysis with One Single-Guide RNA.

CRISPR-based genomic-imaging systems have been utilized for spatiotemporal imaging of the repetitive genomic loci in living cells, but they are still challenged by limited signal-to-noise ratio (SNR) at a non-repetitive genomic locus. Here, an efficient genomic-imaging system is proposed, termed CRISPR/Pepper-tDeg, by engineering the CRISPR sgRNA scaffolds with the degron-binding Pepper aptamers for binding fluorogenic proteins fused with Tat peptide derived degron domain (tDeg). The target-dependent stability switches of both sgRNA and fluorogenic protein allow this system to image repetitive telomeres sensitively with a 5-fold higher SNR than conventional CRISPR/MS2-MCP system using "always-on" fluorescent protein tag. Subsequently, CRISPR/Pepper-tDeg is applied to simultaneously label and track two different genomic loci, telomeres and centromeres, in living cells by combining two systems. Given a further improved SNR by the split fluorescent protein design, CRISPR/Pepper-tDeg system is extended to non-repetitive sequence imaging using only one sgRNA with two aptamer insertions. Neither complex sgRNA design nor difficult plasmid construction is required, greatly reducing the technical barriers to define spatiotemporal organization and dynamics of both repetitive and non-repetitive genomic loci in living cells, and thus demonstrating the large application potential of this genomic-imaging system in biological research, clinical diagnosis and therapy.

Toehold Strand Displacement-Mediated Exponential HCR for Highly Sensitive and Specific Analysis of miRNA in Living Cells.

As an important disease biomarker, the development of sensitive detection strategies for miRNA, especially intracellular miRNA imaging strategies, is helpful for early diagnosis of diseases, pathological research, and drug development. Hybridization chain reaction (HCR) is widely used for miRNA imaging analysis because of its high specificity and lack of biological enzymes. However, the classic HCR reaction exhibits linear amplification with low efficiency, limiting its use for the rapid analysis of trace miRNA in living cells. To address this problem, we proposed a toehold-mediated exponential HCR (TEHCR) to achieve highly sensitive and efficient imaging of miRNA in living cells using ß-FeOOH nanoparticles as transfection vectors. The detection limit of TEHCR was as low as 92.7 fM, which was 8.8 × 103 times lower compared to traditional HCR, and it can effectively distinguish single-base mismatch with high specificity. The TEHCR can also effectively distinguish the different expression levels of miRNA in cancer cells and normal cells. Furthermore, TEHCR can be used to construct OR logic gates for dual miRNA analysis without the need for additional probes, demonstrating high flexibility. This method is expected to play an important role in clinical miRNA-related disease diagnosis and drug development as well as to promote the development of logic gates.

Open and closed microfluidics for biosensing.

Biosensing is vital for many areas like disease diagnosis, infectious disease prevention, and point-of-care monitoring. Microfluidics has been evidenced to be a powerful tool for biosensing via integrating biological detection processes into a palm-size chip. Based on the chip structure, microfluidics has two subdivision types: open microfluidics and closed microfluidics, whose operation methods would be diverse. In this review, we summarize fundamentals, liquid control methods, and applications of open and closed microfluidics separately, point out the bottlenecks, and propose potential directions of microfluidics-based biosensing.

Dual miRNA-Triggered DNA Walker Assisted by APE1 for Specific Recognition of Tumor Cells.

The identification of a specific tumor cell is crucial for the early diagnosis and treatment of cancer. However, it remains a challenge due to the limited sensitivity and accuracy, long response time, and low contrast of the recent approaches. In this study, we develop a dual miRNA-triggered DNA walker (DMTDW) assisted by APE1 for the specific recognition of tumor cells. miR-10b and miR-155 were selected as the research models. Without miR-10b and miR-155 presence, the DNA walker remains inactive as its walking strand of W is locked by L1 and L2. After miR-10b and miR-155 are input, the DNA walker is triggered as miR-10b and miR-155 bind to L1 and L2 of W-L1-L2, respectively, unlocking W. The DNA walker is driven by endogenous APE1 that is highly catalytic and is highly expressed in the cytoplasm of tumor cells but barely expressed in normal cells, ensuring high contrast and reaction efficiency for specific recognition of tumor cells. Dual miRNA input is required to trigger the DNA walker, making this strategy with a high accuracy. The DMTDW strategy exhibited high sensitivity for miRNA analysis with a detection limit of 44.05 pM. Living cell-imaging experiments confirmed that the DMTDW could effectively respond to the fluctuation of miRNA and specifically identified MDA-MB-231 cells from different cell lines. The proposed DMTDW is sensitive, rapid, and accurate for specific tumor cell recognition. We believe that the DMTDW strategy can become a powerful diagnostic tool for the specific recognition of tumor cells.

MiRNA and mRNA-Controlled Double-Cascaded Amplifying Circuit Nanosensor for Accurate Discrimination of Breast Cancers in Living Cells, Animals, and Organoids.

Metastasis is the leading cause of death in patients with breast cancer. Detecting high-risk breast cancer, including micrometastasis, at an early stage is vital for customizing the right and efficient therapies. In this study, we propose an enzyme-free isothermal cascade amplification-based DNA logic circuit in situ biomineralization nanosensor, HDNAzyme@ZIF-8, for simultaneous imaging of multidimensional biomarkers in live cells. Taking miR-21 and Ki-67 mRNA as the dual detection targets achieved sensitive logic operations and molecular recognition through the cascade hybridization chain reaction and DNAzyme. The HDNAzyme@ZIF-8 nanosensor has the ability to accurately differentiate breast cancer cells and their subtypes by comparing their relative fluorescence intensities. Of note, our nanosensor can also achieve visualization within breast cancer organoids, faithfully recapitulating the functional characteristics of parental tumor. Overall, the combination of these techniques offers a universal strategy for detecting cancers with high sensitivity and holds vast potential in clinical cancer diagnosis.

Identifying Protein Phosphorylation Site-Disease Associations Based on Multi-Similarity Fusion and Negative Sample Selection by Convolutional Neural Network.

As one of the most important post-translational modifications (PTMs), protein phosphorylation plays a key role in a variety of biological processes. Many studies have shown that protein phosphorylation is associated with various human diseases. Therefore, identifying protein phosphorylation site-disease associations can help to elucidate the pathogenesis of disease and discover new drug targets. Networks of sequence similarity and Gaussian interaction profile kernel similarity were constructed for phosphorylation sites, as well as networks of disease semantic similarity, disease symptom similarity and Gaussian interaction profile kernel similarity were constructed for diseases. To effectively combine different phosphorylation sites and disease similarity information, random walk with restart algorithm was used to obtain the topology information of the network. Then, the diffusion component analysis method was utilized to obtain the comprehensive phosphorylation site similarity and disease similarity. Meanwhile, the reliable negative samples were screened based on the Euclidean distance method. Finally, a convolutional neural network (CNN) model was constructed to identify potential associations between phosphorylation sites and diseases. Based on tenfold cross-validation, the evaluation indicators were obtained including accuracy of 93.48%, specificity of 96.82%, sensitivity of 90.15%, precision of 96.62%, Matthew's correlation coefficient of 0.8719, area under the receiver operating characteristic curve of 0.9786 and area under the precision-recall curve of 0.9836. Additionally, most of the top 20 predicted disease-related phosphorylation sites (19/20 for Alzheimer's disease; 20/16 for neuroblastoma) were verified by literatures and databases. These results show that the proposed method has an outstanding prediction performance and a high practical value.

A dual-signal amplification strategy based on rolling circle amplification and APE1-assisted amplification for highly sensitive and specific miRNA analysis for early diagnosis of alzheimer's disease.

MicroRNA (miRNA) is involved in the progression of Alzheimer's disease (AD) and emerges as a promising AD biomarker and therapeutic target. Therefore, there is an urgent need to develop convenient and precise miRNA detection methods for AD diagnosis. Herein, a dual-signal amplification strategy based on rolling circle amplification and APE1-assisted amplification for miRNA analysis for early diagnosis of AD was proposed. The strategy consisted of dumbbell-shaped probe (DP) as amplification template and a reporter probe (RP) with an AP site modification. In the presence of the target miRNA, the miRNAs bound to the toehold domain of DP and DP was activated into a circular template. Then, RCA reaction was triggered, producing a large number of long-stranded products containing repeated sequences. After RCA, APE1 enzyme recognized and removed AP site in the complex of RCA/RP products. By coupling RCA with APE1-assisted amplification, this method has high sensitivity with the limit of detection (LOD) of 1.82 fM. Moreover, by using DP as template for RCA reaction, high specificity can be achieved. By detecting miR-206 in serum using this method, the expression of miR-206 can be accurately distinguished between AD patients and healthy individuals, indicating that this method has broad application prospects in clinical diagnosis.

Label-free fluorescence detection of human 8-oxoguanine DNA glycosylase activity amplified by target-induced rolling circle amplification.

BACKGROUND: Human 8-oxoG DNA glycosylase 1 (hOGG1) is one of the important members of DNA glycosylase for Base excision repair (BER), the abnormal activity of which can lead to the failure of BER and the appearance of various diseases, such as breast cancer, bladder cancer, Parkinson's disease and lung cancer. Therefore, it is important to detect the activity of hOGG1. However, traditional detection methods suffer from time consuming, complicated operation, high false positive results and low sensitivity. Thus, it remains a challenge to develop simple and sensitive hOGG1 analysis strategies to facilitate early diagnosis and treatment of the relative disease. RESULTS: A target-induced rolling circle amplification (TIRCA) strategy for label-free fluorescence detection of hOGG1 activity was proposed with high sensitivity and specificity. The TIRCA strategy was constructed by a hairpin probe (HP) containing 8-oxoG site and a primer probe (PP). In the presence of hOGG1, the HP transformed into dumbbell DNA probe (DDP) after the 8-oxoG site of which was removed. Then the DDP formed closed circular dumbbell probe (CCDP) by ligase. CCDP could be used as amplification template of RCA to trigger RCA. The RCA products containing repeated G4 sequences could combine with ThT to produce enhanced fluorescence, achieving label-free fluorescence sensing of hOGG1. Given the high amplification efficiency of RCA and the high fluorescence quantum yield of the G4/ThT, the proposed TIRCA achieved highly sensitive measurement of hOGG1 activity with a detection limit of 0.00143 U/mL. The TIRCA strategy also exhibited excellent specificity for hOGG1 analysis over other interference enzymes. SIGNIFICANCE: This novel TIRCA strategy demonstrates high sensitivity and high specificity for the detection of hOGG1, which has also been successfully used for the screening of inhibitors and the analysis of hOGG1 in real samples. We believe that this TIRCA strategy provides new insight into the use of the isothermal nucleic acid amplification as a useful tool for hOGG1 detection and will play an important role in disease early diagnosis and treatment.

A Simple and Robust Exponential Amplification Reaction (EXPAR)-Based Hairpin Template (exp-Hairpin) for Highly Specific, Sensitive, and Universal MicroRNA Detection.

Specific and sensitive detection of microRNAs continues to encounter significant challenges, especially in the development of rapid and efficient isothermal amplification strategies for point-of-care settings. The exponential amplification reaction (EXPAR) has garnered significant attention owing to its simplicity and rapid amplification of signals within a short period. However, a substantial loss of amplification efficiency, difficulty in distinguishing closely related homologous sequences, and adapting the designed templates to other targets seriously hamper the practical application of the EXPAR. In this work, a hairpin template tailored for the EXPAR system (exp-Hairpin) was constructed by adding identical trigger sequences and enzyme cleavage sites on two arms of the hairpin, achieving theoretically more than 2n amplification efficiency and minimal background amplification of EXPAR. Modulating the stability of the exp-Hairpin template by increasing the stem length, the specificity of detecting target miRNA in highly homologous sequences could be significantly improved. Using miRNA let-7a as a target model, the exp-Hairpin with 8 bp stem length for EXPAR amplification curves could effectively distinguish target let-7a and nontarget let-7b/7c/7f/7g/7i homologous sequences. This strategy enabled the sensitive and accurate analysis of let-7a in diluted human serum with satisfactory recoveries. By simply replacing the loop recognition sequence of exp-Hairpin, the specific detection of miR-200b was also achieved, demonstrating the universality of this strategy. The exp-Hairpin EXPAR accelerates simple and rapid molecular diagnostic applications for short nucleic acids.

Dual-Signal Amplification Strategy Based on Catalytic Hairpin Assembly and APE1-Assisted Amplification for High-Contrast miRNA Imaging in Living Cells.

Early tumor diagnosis is crucial to successful treatment. Earlier studies have shown that microRNA is a biomarker for early tumor diagnosis. The development of highly sensitive miRNA detection methods, especially in living cells, plays an indispensable role for early diagnosis and treatment of tumor. Although the catalytic hairpin assembly (CHA)-based miRNA analysis strategy is commonly used for disease diagnosis, further application of CHA is hindered due to its low amplification efficiency and low tumor recognition contrast. To address these limitations, we propose a dual-signal amplification strategy based on CHA and APE1-assisted amplification, enabling highly sensitive and high-contrast miRNA imaging. The miR-221 was selected as a target model. This dual-signal amplification strategy has exhibited high amplification efficiency, which could analyze miRNA as low as 21 fM. This strategy also exhibited high specificity, which could distinguish target miRNA and nontarget with single-base differences. Moreover, this method showed significant potential for practical application, as it could successfully distinguish the expression difference of miR-221 in the plasma samples of normal people and patients. Most importantly, the expression level of the APE1 enzyme in tumor cells is higher than that in normal cells, allowing this strategy to sensitively and specifically image miRNA within tumor cells. This proposed method has also been successfully used to indicate fluctuations of intracellular miRNA and to distinguish miRNA expression between normal cells and cancer cells with high contrast. We anticipate that this method will provide fresh insights and can be a powerful tool for tumor diagnosis and treatment based on miRNA analysis.

A binary system based DNA tetrahedron and fluorogenic RNA aptamers for highly specific and label-free mRNA imaging in living cells.

Developing simple, rapid and specific mRNA imaging strategy plays an important role in the early diagnosis of cancer and the new drugs development. Herein, we have established a novel binary system based DNA tetrahedron and fluorogenic RNA aptamers for highly specific and label-free mRNA imaging in living cells. This developed system consisted of tetrahedron probe A (TPA) and tetrahedron probe B (TPB). TK1 mRNA was chosen as the study model. After TPA and TPB enter into the live cells, the TK1 mRNA induces TPA and TPB to approach and activate the fluorescent aptamer, resulting in enhanced fluorescent signal in the presence of small molecules of DFHBI-1T. By this design, the high specificity label-free detection of nucleic acids was achieved with a detection limit of 1.34 nM. Confocal fluorescence imaging experiments had proved that this strategy could effectively distinguish the TK1 mRNA expression level between normal cell and cancer cell. The developed method is expected to provide a new tool for early diagnosis of diseases and new drug development.

Dephosphorylation Switch DNAzyme-RCA Circuit: A Robust Strategy for the Homogeneous and Reliable Detection of FTO Demethylase.

Fat mass and obesity-associated protein (FTO) plays a crucial role in regulating the dynamic modification of N6-methyladenosine (m6A) in eukaryotic mRNA. Sensitive detection of the FTO level and efficient evaluation of the FTO demethylase activity are of great importance to early cancer diagnosis and anticancer drug discovery, which are currently challenged by limited sensitivity/precision and low throughput. Herein, a robust strategy based on the dephosphorylation switch DNAzyme-rolling circle amplification (RCA) circuit, termed DSD-RCA, is developed for highly sensitive detection of FTO and inhibitor screening. Initially, the catalytic activity of DNAzyme is silenced by engineering with an m6A modification in its catalytic core. Only in the presence of target FTO can the methyl group on DNAzyme be eliminated, resulting in the activation of the catalytic activity of DNAzyme and thus cleaving the hairpin substrate to release numerous primers. Different from the conventional methods that use the downstream cleavage primer with the original 3'-hydroxyl end directly as the RCA primer with the problem of high background signal, which should be compensated by additional separation and wash steps in heterogeneous format, our DSD-RCA assay uses the upstream cleavage primer with a 2',3'-cyclic phosphate terminus at the 3'-end serving as an intrinsically blocked 3' end. Only after a dephosphorylation reaction mediated by T4 polynucleotide kinase can the upstream cleavage primers with a resultant 3'-hydroxyl end be extended by RCA. With the high signal-to-noise ratio and homogeneous property, the proposed platform can sensitively detect FTO with a limit of detection of 31.4 pM, and the relative standard deviations (RSDs %) ranging from 0.8 to 2.0% were much lower than the heterogeneous methods. The DSD-RCA method was applied for analyzing FTO in cytoplasmic lysates from different cell lines and tissues of breast cancer patients and further used for screening FTO inhibitors without the need for separation or cleaning, providing an opportunity for achieving high throughput and demonstrating the potential applications of this strategy in disease diagnostics, drug discovery, and biological applications.

A DNAzyme dual-feedback autocatalytic exponential amplification biocircuit for microRNA imaging in living cells.

Autocatalytic biocircuit are powerful tools for analysing intracellular biomarkers, but these tools are constrained by limitations in amplification capacity and intracellular delivery efficiency. In this work, we developed a DNAzyme-based dual-feedback autocatalytic exponential amplification biocircuit sustained by a honeycomb MnO2 nanosponge (EDA2@hMNS) for live-cell imaging of intracellular low-abundance microRNAs (miRNA). The EDA2 biocircuit comprises a blocked DNAzyme (b-DNAzyme), a Fuel strand and a Substrate strand. In the EDA2 biocircuit, target miRNAs are recycled and feedback for rounds of DNAzymatic amplification, and the DNAzymatic reactions continuously generate target miRNA analogues for dual-feedback to achieve multiple parallel cascade DNAzymatic reactions that improve amplification capacity substantially. In addition, the hMNS ensures high loading and delivery efficiency of biocircuit probes into living cells and also provides sufficient Mn2+ DNAzyme cofactor from in situ decomposition by intracellular glutathione (GSH). The EDA2@hMNS realized a detection limit of 17 pM, which is 288-fold lower than the b-DNAzyme lacking the DNAzymatic amplification. These results demonstrate the great promise for this critical tool in analysing low-abundance biomarkers and cancer diagnostics.

On-Site-Activated Transmembrane Logic DNA Nanodevice Enables Highly Specific Imaging of Cancer Cells by Targeting Tumor-Related Nucleolin and Intracellular MicroRNA.

The ability to specifically image cancer cells is essential for cancer diagnosis; however, this ability is limited by the false positive associated with single-biomarker sensors and off-site activation of "always active" nucleic acid probes. Herein, we propose an on-site, activatable, transmembrane logic DNA (TLD) nanodevice that enables dual-biomarker sensing of tumor-related nucleolin and intracellular microRNA for highly specific cancer cell imaging. The TLD nanodevice is constructed by assembling a tetrahedral DNA nanostructure containing a linker (L)-blocker (B)-DNAzyme (D)-substrate (S) unit. AS-apt, a DNA strand containing an elongated segment and the AS1411 aptamer, is pre-anchored to nucleolin protein, which is specifically expressed on the membrane of cancer cells. Initially, the TLD nanodevice is firmly sealed by the blocker containing an AS-apt recognition zone, which prevents off-site activation. When the nanodevice encounters a target cancer cell, AS-apt (input 1) binds to the blocker and unlocks the sensing ability of the nanodevice for miR-21 (input 2). The TLD nanodevice achieves dual-biomarker sensing from the cell membrane to the cytoplasm, thereby ensuring cancer cell-specific imaging. This TLD nanodevice represents a promising strategy for the highly reliable analysis of intracellular biomarkers and a promising platform for cancer diagnosis and related biomedical applications.

Endogenous Enzyme-Powered DNA Nanomotor Operating in Living Cells for microRNA Imaging.

Accurate and specific imaging of low-abundance microRNA (miRNA) in living cells is extremely important for disease diagnosis and monitoring of disease progression. DNA nanomotors have shown great potential for imaging molecules of interest in living cells. However, inappropriate driving forces and complex design and operation procedures have hindered their further application. Here, we proposed an endogenous enzyme-powered DNA nanomotor (EEPDN), which employs an endogenous APE1 enzyme as fuel to execute repetitive cycles of motion for miRNA imaging in living cells. The whole motor system is constructed based on gold nanoparticles without other auxiliary additives. Due to the high efficiency of APE1, this EEPDN system has achieved highly sensitive miRNA imaging in living cells within 1.5 h. This strategy was also successfully used to differentiate the expression of specific miRNA between tumor cells and normal cells, demonstrating a high tumor cell selectivity. This strategy can promote the development of novel nanomotors and is expected to be a perfect intracellular molecular imaging tool for biological and medical applications.

DNAzyme-Amplified Cascade Catalytic Hairpin Assembly Nanosystem for Sensitive MicroRNA Imaging in Living Cells.

Sensitive imaging of microRNAs (miRNAs) in living cells is significant for accurate cancer clinical diagnosis and prognosis research studies, but it is challenged by inefficient intracellular delivery, instability of nucleic acid probes, and limited amplification efficiency. Herein, we engineered a DNAzyme-amplified cascade catalytic hairpin assembly (CHA)-based nanosystem (DCC) that overcomes these challenges and improves the imaging sensitivity. This enzyme-free amplification nanosystem is based on the sequential activation of DNAzyme amplification and CHA. MnO2 nanosheets were used as nanocarriers for the delivery of nucleic acid probes, which can resist the degradation by nucleases and supply Mn2+ for the DNAzyme reaction. After entering into living cells, the MnO2 nanosheets can be decomposed by intracellular glutathione (GSH) and release the loaded nucleic acid probes. In the presence of target miRNA, the locking strand (L) was hybridized with target miRNA, and the DNAzyme was released, which then cleaved the substrate hairpin (H1). This cleavage reaction resulted in the formation of a trigger sequence (TS) that can activate CHA and recover the fluorescence readout. Meanwhile, the DNAzyme was released from the cleaved H1 and bound to other H1 for new rounds of DNAzyme-based amplification. The TS was also released from CHA and involved in the new cycle of CHA. By this DCC nanosystem, low-abundance target miRNA can activate many DNAzyme and generate numerous TS for CHA, resulting in sensitive and selective analysis of miRNAs with a limit of detection of 5.4 pM, which is 18-fold lower than that of the traditional CHA system. This stable, sensitive, and selective nanosystem holds great potential for miRNA analysis, clinical diagnosis, and other related biomedical applications.

Care patterns and Traditional Chinese Medicine constitution as factors of depression and anxiety in patients with systemic sclerosis: A cross-sectional study during the COVID-19 pandemic.

Objective: Care patterns and Traditional Chinese Medicine (TCM) constitution affects the emotion and health of patients with systemic sclerosis (SSc) while the prevalence of COVID-19 may aggravate such patients' emotion and health. We investigated the depression and anxiety levels of patients with SSc during the pandemic to identify the correlation between care patterns, TCM constitution, and patients' emotion. Materials and methods: This was a cross-sectional study. Patients with SSc and healthy individuals were surveyed using the patient health questionnaire-9, generalized anxiety disorder-7, and constitution in Chinese medicine questionnaire and a modified care pattern questionnaire. Factors correlated with depression and anxiety were screened using univariate and multivariate logistic regression analyses. Results: A total of 273 patients with SSc and 111 healthy individuals were included in the analysis. The proportion of patients with SSc who were depressed was 74.36%, who had anxiety was 51.65%, and who experienced disease progression during the pandemic was 36.99%. The proportion of income reduction in the online group (56.19%) was higher than that in the hospital group (33.33%) (P = 0.001). Qi-deficiency [adjusted odds ratio (OR) = 2.250] and Qi-stagnation (adjusted OR = 3.824) constitutions were significantly associated with depression. Remote work during the outbreak (adjusted OR = 1.920), decrease in income (adjusted OR = 3.556), and disease progression (P = 0.030) were associated with the occurrence of depression. Conclusion: Chinese patients with SSc have a high prevalence of depression and anxiety. The COVID-19 pandemic has changed the care patterns of Chinese patients with SSc, and work, income, disease progression, and change of medications were correlates of depression or anxiety in patients with SSc. Qi-stagnation and Qi-deficiency constitutions were associated with depression, and Qi-stagnation constitution was associated with anxiety in patients with SSc. Trial registration: http://www.chictr.org.cn/showproj.aspx?proj=62301, identifier ChiCTR2000038796.

Isolation of Structurally Heterogeneous TCR-CD3 Extracellular Vesicle Subpopulations Using Caliper Strategy.

Cells in different states can release diverse types of extracellular vesicles (EVs) that participate in intracellular communication or pathological processes. The identification and isolation of EV subpopulations are significant to explore their physiological functions and clinical value. In this study, structurally heterogeneous T-cell receptor (TCR)-CD3 EVs were proposed and verified for the first time using a caliper strategy. Two CD3-targeting aptamers were designed in the shape of a caliper with an optimized probe distance and were assembled on gold nanoparticles (Au-Caliper) to distinguish TCR-CD3 monomeric and dimeric EVs (m/dCD3 EVs) in skin-transplanted mouse plasma. Phenotyping and sequencing analysis revealed clear heterogeneity in the isolated m/dCD3 EVs, providing the potential for mCD3 EVs as a candidate biomarker of acute cellular rejection (ACR) and holding great prospects for distinguishing EV subpopulations based on protein oligomerization states.