Mediating role of inflammatory indicators in the association between sleep status and blood pressure in centenarians: evidence from China Hainan Centenarian Cohort Study.

Objectives: To conduct a comprehensive analysis in Hainan centenarians on the link between sleep status and their blood pressure status. Furthermore, the study also aims to explore how inflammatory indicators may mediate the relationship. Methods: The China Hainan Centenarians Cohort Study (CHCCS) collected baseline data on sleep status, inflammatory indicators, and blood pressure data. The study used a mediation model to investigate how inflammatory indicators mediate the relationship between sleep status and blood pressure status. Result: In this study, a total of 967 centenarians were included. The prevalence of hypertension among the centenarians was 71.4%. The analysis showed that centenarians with poor sleep quality had a 43% higher risk of hypertension compared to those with normal sleep quality (OR = 1.43, 95% CI: 1.03-1.97). Additionally, centenarians with nighttime sleep durations of ≤ 6 h or > 9 h had higher proportions of high pulse pressure (PP), with OR values of 1.76 (95% CI: 1.18-2.63) and 2.07 (95% CI: 1.34-3.19), respectively. Mediation analysis illustrated that complement C3 played a mediating role in the relationship between sleep quality and hypertension, with an effect ratio of 2.4%. Similarly, lymphocyte count, the neutrophil-to-lymphocyte ratio (NLR), and the systemic immune-inflammation index (SII) were identified as mediating factors in the association between nighttime sleep duration and high PP, with effect ratios of 91.22%, 36.93%, and 0.20%, respectively. Conclusion: In centenarians, poor sleep quality raises the risk of hypertension, with complement C3 as a mediator. Additionally, nighttime sleep durations of ≤ 6 h or > 9 h increases the risk of high PP, mediated by lymphocyte count, NLR, and SII.

Identification of common and specific cold resistance pathways from cold tolerant and non-cold tolerant mango varieties.

Mango has frequently encountered severe climate and environmental challenges such as low temperatures, seriously affecting the sustainable development of the industry. In the study, physiological measurements showed that the activities of superoxide dismutase (SOD) and peroxidase (POD) were found to be higher in Jinhuang (JH) mango plants than those of Tainong (TN) mango plants under cold stress, indicating cold tolerant (JH) and non-cold tolerant (TN) mango varieties were firstly determined. Subsequently, transcriptomics showed 8,337 and 7,996 differentially expressed genes (DEGs) were respectively identified in JH and TN mango varieties treated at 4 °C for 36 h, while more DEGs (10,683 and 10,723) were screened when treated at 4 °C for 72 h. Quantitative real-time PCR (qRT-PCR) of the selected DEGs confirmed their transcriptional levels displayed agreement to the transcriptome data. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed two primary cold resistant regulation pathways, photosynthesis-antenna proteins pathway and photosynthesis pathway, were both significant annotated in the two mango varieties, indicating share the common regulation mechanism response to cold stress. However, five specific cold resistant pathways, such as amino acid and carbohydrate metabolisms, were identified in JH mango variety with cold stress for longer duration, indicating the specific regulation pathways in the cold tolerant mango varieties. Furthermore, 43 ethylene-responsive transcription factors (ERFs) were significantly annotated in JH mango after cold-treated for 72 h comparing with the control group, and three of them ERF109-1, ERF017-1 and ERF017-2 were highly expressed, which may play important regulatory roles in plant cold resistance. These results provided insights into the primary and specific molecular mechanisms of different mango varieties resistance to chill.

Inertial and Deterministic Lateral Displacement Integrated Microfluidic Chips for Epithelial-Mesenchymal Transition Analysis.

With the aim of efficiently sorting rare circulating tumor cells (CTCs) from blood and minimizing damage to CTCs during isolation, we constructed an inertia-assisted single-cell focusing generator (I-SCF) and a water droplet deterministic lateral displacement cell sorting (D-DLD) microfluidic system (IDIC) based on different sizes, the device is initially sorted by a continuous fluid swing and Dean flow-assisted helical micromixers, then flows through a droplet shaped DLD region, enabling single-cell focused sequencing and precise separation, improving cell separation efficiency (>95%) and purity, while ensuring a high single cells survival rate of more than 98.6%. Subsequently, breast cancer cell lines were run through our chip, and then the downstream epithelial-mesenchymal transition (EMT) process induced by TGF-ß was detected, and the levels of three proteins, EpCAM, PD-L1, and N-cadherin, were analyzed to establish the relationship between PD-L1 and the EMT process. Compared with other analytical techniques such as the filtration method, the enrichment method and immunoaffinity capture methods, this method not only ensures the separation efficiency and purity, but also ensures the cell activity, and avoids missing the different results caused by the heterogeneity of CTCs due to the isolation of high purity (84.01%). The device has a high throughput processing capacity (5 mL of diluted whole blood/∼2.8 h). By using the chip, we can more easily and conveniently predict tumor stage and carry out cancer prevention and treatment in advance, and it is expected to be further developed into a clinical liquid biopsy technology in the future.

Composition Tuning of Semi-Open Cell Carriers via Phase Freeze-Shrink Self-Molding.

Extracellular matrix (ECM)-mimicking microsized cell carriers featuring a semi-isolated chamber facilitate the study of cellular heterogeneity as well as intercellular communication. However, the semiopen shaping of the designated gel mixture remains unattainable with current methods. We report an oil-phase freeze-shrink self-molding mechanism for generating size- and composition-tunable cradle-shaped microgels (microcradles) from water-in-oil droplets. The universality of this shape transition principle is demonstrated with six types of polysaccharides dispersed in a poly(ethylene glycol) diacrylate (PEGDA) or methacrylate gelatin (GelMA) matrix. By doping the microcradles with the major ECM component, hyaluronic acid sodium, we demonstrate a label-free selective culture of CD44 receptor-rich cells and the formation of cell spheroids within 3 days. This cryo-induced cradle-shaping strategy enables the functionalization of microcarriers for selective cell culture, thereby allowing them to be used for intercellular communication, drug delivery, and the construction of structural units for osteogenesis and 3D printing.

CRISPR-based dual-aptamer proximity ligation coupled hybridization chain reaction for precise detection of tumor extracellular vesicles and cancer diagnosis.

Tumor cell-derived extracellular vesicles (TEVs) contain numerous cellular molecules and are considered potential biomarkers for non-invasive liquid biopsy. However, due to the low abundance of TEVs secreted by tumor cells and their phenotypic heterogeneity, there is a lack of sensitive and specific methods to quantify TEVs. Here, we developed a dual-aptamer proximity ligation-coupled hybridization chain reaction (HCR) method for tracing TEVs, exploiting CRISPR to achieve highly sensitive detection. Taking advantage of the high binding affinity of aptamers, the two aptamers (AptEpCAM, AptHER2) exhibited the high selectivity for TEVs recognition. HCR generated long-repeated sequence containing multiple crRNA targetable barcodes, and the signals were further amplified by CRISPR upon recognizing the HCR sequences, thereby enhancing the sensitivity. Under optimal conditions, the developed method demonstrated a favorable linear relationship in the range of 2 × 103-107 particles/µL, with a limit of detection (LOD) of 3.3 × 102 particles/µL. We directly applied our assay to clinical plasma analysis, achieving 100 % accuracy in cancer diagnosis, thus demonstrating the potential clinical applications of TEVs. Due to its simplicity and rapidity, excellent sensitivity and specificity, this method has broad applications in clinical medicine.

Multiplex Sensing of Biomarkers on the Cancer Cell Surface by an Epithelial-Mesenchymal Transition (EMT) Sensing Panel Enables Precise Differentiating of Cancer Cells at Various EMT Stages.

Epithelial-mesenchymal transition (EMT) is a complex process that plays a critical role in tumor progression. In this study, we present an EMT sensing panel for the classification of cancer cells at different EMT stages. This sensing panel consists of three types of fluorescent probes based on boronic acid-functionalized carbon-nitride nanosheet (BCN) derivatives. The selective response toward different EMT-associated biomarkers, namely, EpCAM, N-cadherin, and sialic acid (SA), was achieved by conjugating the corresponding antibodies to each BCN derivative, whereas the rare-earth-doping ensures simultaneous sensing of the three biomarkers with fluorescent emission of the three probes at different wavelengths. Sensitive sensing of the three biomarkers was achieved at the protein level with LODs reaching 1.35 ng mL-1 for EpCAM, 1.62 ng mL-1 for N-cadherin, and 1.54 ng mL-1 for SA. The selective response of these biomarkers on the cell surface also facilitated sensitive detection of MCF-7 cells and MDA-MB-231 cells with LODs of 2 cells/mL and 2 cells/mL, respectively. Based on the simultaneous sensing of the three biomarkers on cancer cells that underwent different extents of EMT, precise discrimination and classification of cells at various EMT stages were also achieved with an accuracy of 93.3%. This EMT sensing panel provided a versatile tool for monitoring the EMT evolution process and has the potential to be used for the evaluation of the EMT-targeting therapy and metastasis prediction.

Portable Photoacoustic Analytical System Combined with Wearable Hydrogel Patch for pH Monitoring in Chronic Wounds.

Timely diagnosis, monitoring, and management of chronic wounds play crucial roles in improving patients' quality of life, but clinical evaluation of chronic wounds is still ambiguous and relies heavily on the experience of clinician, resulting in increased social and financial burden and delay of optimal treatment. During the different stages of the healing process, specific and dynamic changes of pH values in the wound exudate can be used as biomarkers to reflect the wound status. Herein, a pH-responsive agent with well-behaved photoacoustic (PA) properties, nitrazine yellow (NY), was incorporated in poly(vinyl alcohol)/sucrose (PVA/Suc) hydrogel to construct a wearable pH-sensing patch (PVA/Suc/NY hydrogel) for monitoring of pH values during chronic wound healing. According to Rosencwaig-Gersho theory and the combination of 3D printing technology, the PA chamber volume and chopping frequency were systematically optimized to improve the sensitivity of the PA analytical system. The prepared PVA/Suc/NY hydrogel patch had excellent mechanical properties and flexibility and could maintain conformal contact with skin. Moreover, combined with the miniaturized PA analytical device, it had the potential to detect pH values (5.0-9.0) free from the color interference of blood and therapeutic drugs, which provides a valuable strategy for wound pH value monitoring by PA quantitation. This strategy of combining the wearable hydrogel patch with portable PA analysis offers broad new prospects for the treatment and management of chronic wounds due to its features of simple operation, time savings, and anti-interference.

Microglial AKAP8L: a key mediator in diabetes-associated cognitive impairment via autophagy inhibition and neuroinflammation triggering.

BACKGROUND: Diabetes-associated cognitive impairment (DACI) poses a significant challenge to the self-management of diabetes, markedly elevating the risk of adverse complications. A burgeoning body of evidence implicates microglia as a central player in the pathogenesis of DACI. METHODS: We utilized proteomics to identify potential biomarkers in high glucose (HG)-treated microglia, followed by gene knockdown techniques for mechanistic validation in vitro and in vivo. RESULTS: Our proteomic analysis identified a significant upregulation of AKAP8L in HG-treated microglia, with concurrent dysregulation of autophagy and inflammation markers, making AKAP8L a novel biomarker of interest. Notably, the accumulation of AKAP8L was specific to HG-treated microglia, with no observed changes in co-cultured astrocytes or neurons, a pattern that was mirrored in streptozotocin (STZ)-induced diabetic mice. Further studies through co-immunoprecipitation and proximity ligation assay indicated that the elevated AKAP8L in HG-treated microglial cells interacts with the mTORC1. In the STZ mouse model, we demonstrated that both AKAP8L knockdown and rapamycin treatment significantly enhanced cognitive function, as evidenced by improved performance in the Morris water maze, and reduced microglial activation. Moreover, these interventions effectively suppressed mTORC1 signaling, normalized autophagic flux, mitigated neuroinflammation, and decreased pyroptosis. CONCLUSIONS: Our findings highlight the critical role of AKAP8L in the development of DACI. By interacting with mTORC1, AKAP8L appears to obstruct autophagic processes and initiate a cascade of neuroinflammatory responses. The identification of AKAP8L as a key mediator in DACI opens up new avenues for potential therapeutic interventions.

Effects of respiratory muscle training on post-stroke rehabilitation: A systematic review and meta-analysis.

BACKGROUND: Stroke often results in significant respiratory dysfunction in patients. Respiratory muscle training (RMT) has been proposed as a rehabilitative intervention to address these challenges, but its effectiveness compared to routine training remains debated. This systematic review and meta-analysis aim to evaluate the effects of RMT on exercise tolerance, muscle strength, and pulmonary function in post-stroke patients. AIM: To systematically assess the efficacy of RMT in improving exercise tolerance, respiratory muscle strength, and pulmonary function in patients recovering from a stroke, and to evaluate whether RMT offers a significant advantage over routine training modalities in enhancing these critical health outcomes in the post-stroke population. METHODS: Following the Preferred Reporting Items for Systematic reviews and Meta-Analyses guidelines, a comprehensive search across PubMed, Embase, Web of Science, and the Cochrane Library was conducted on October 19, 2023, without temporal restrictions. Studies were selected based on the predefined inclusion and exclusion criteria focusing on various forms of RMT, control groups, and outcome measures [including forced expiratory volume in the first second (FEV1), forced vital capacity (FVC), maximal voluntary ventilation (MVV), peak expiratory flow (PEF), maximal inspiratory pressure (MIP), maximal expiratory pressure (MEP), and 6-min walking test (6MWT)]. Only randomized controlled trials (RCTs) were included. Data extraction and quality assessment were conducted independently by two reviewers using the Cochrane Collaboration's risk of bias tool. Statistical analyses, including those using the fixed-effect and random-effects models, sensitivity analysis, and publication bias assessment, were performed using Review Manager software. RESULTS: A total of 15 RCTs were included. Results indicated significant improvements in MIP (12.51 cmH2O increase), MEP (6.24 cmH2O increase), and various pulmonary function parameters (including FEV1, FVC, MVV, and PEF). A substantial increase in 6MWT distance (22.26 meters) was also noted. However, the heterogeneity among studies was variable, and no significant publication bias was detected. CONCLUSION: RMT significantly enhances walking ability, respiratory muscle strength (MIP and MEP), and key pulmonary function parameters (FEV1, FVC, MVV, and PEF) in post-stroke patients. These findings support the incorporation of RMT into post-stroke rehabilitative protocols.

Harnessing virus flexibility to selectively capture and profile rare circulating target cells for precise cancer subtyping.

The effective isolation of rare target cells, such as circulating tumor cells, from whole blood is still challenging due to the lack of a capturing surface with strong target-binding affinity and non-target-cell resistance. Here we present a solution leveraging the flexibility of bacterial virus (phage) nanofibers with their sidewalls displaying target circulating tumor cell-specific aptamers and their ends tethered to magnetic beads. Such flexible phages, with low stiffness and Young's modulus, can twist and adapt to recognize the cell receptors, energetically enhancing target cell capturing and entropically discouraging non-target cells (white blood cells) adsorption. The magnetic beads with flexible phages can isolate and count target cells with significant increase in cell affinity and reduction in non-target cell absorption compared to magnetic beads having rigid phages. This differentiates breast cancer patients and healthy donors, with impressive area under the curve (0.991) at the optimal detection threshold (>4 target cells mL-1). Immunostaining of captured circulating tumor cells precisely determines breast cancer subtypes with a diagnostic accuracy of 91.07%. Our study reveals the power of viral mechanical attributes in designing surfaces with superior target binding and non-target anti-fouling.

Ultrasensitive and Wash-Free Detection of Tumor Extracellular Vesicles by Aptamer-Proximity-Ligation-Activated Rolling Circle Amplification Coupled to Single Particle ICP-MS.

Tumor-derived extracellular vesicles (TEVs) are rich in cellular information and hold great promise as a biomarker for noninvasive cancer diagnosis. However, accurate measurement of TEVs presents challenges due to their low abundance and potential interference from a high number of EVs derived from normal cells. Herein, an aptamer-proximity-ligation-activated rolling circle amplification (RCA) method for EV membrane recognition, coupled with single particle inductively coupled plasma mass spectrometry (sp-ICP-MS) for the quantification of TEVs, is developed. When DNA-labeled ultrasmall gold nanoparticle (AuNP) probes bind to the long chains formed by RCA, they aggregate to form large particles. Notably, small AuNPs scarcely produce pulse signals in sp-ICP-MS, thereby detecting TEVs in a wash-free manner. By leveraging the strong binding affinity of aptamers, dual aptamers for EpCAM and PD-L1 recognition, and the sp-ICP-MS technique, this method offers remarkable sensitivity and selectivity in tracing TEVs. Under optimized conditions, the present method shows a favorable linear relationship between the pulse signal frequency of sp-ICP-MS and TEV concentration within the range of 105-107 particles/mL, along with a detection limit of 1.1 × 104 particles/mL. The pulse signals from sp-ICP-MS combined with machine learning algorithms are used to discriminate cancer patients from healthy donors with 100% accuracy. Due to its simple and fast operation and excellent sensitivity and accuracy, this approach holds significant potential for diverse applications in life sciences and personalized medicine.

Multispectral 3D DNA Machine Combined with Multimodal Machine Learning for Noninvasive Precise Diagnosis of Bladder Cancer.

Extracellular vesicle (EV) molecular phenotyping offers enormous opportunities for cancer diagnostics. However, the majority of the associated studies adopted biomarker-based unimodal analysis to achieve cancer diagnosis, which has high false positives and low precision. Herein, we report a multimodal platform for the high-precision diagnosis of bladder cancer (BCa) through a multispectral 3D DNA machine in combination with a multimodal machine learning (ML) algorithm. The DNA machine was constructed using magnetic microparticles (MNPs) functionalized with aptamers that specifically identify the target of interest, i.e., five protein markers on bladder-cancer-derived urinary EVs (uEVs). The aptamers were hybridized with DNA-stabilized silver nanoclusters (DNA/AgNCs) and a G-quadruplex/hemin complex to form a sensing module. Such a DNA machine ensured multispectral detection of protein markers by fluorescence (FL), inductively coupled plasma mass spectrometry (ICP-MS), and UV-vis absorption (Abs). The obtained data sets then underwent uni- or multimodal ML for BCa diagnosis to compare the analytical performance. In this study, urine samples were obtained from our prospective cohort (n = 45). Our analytical results showed that the 3D DNA machine provided a detection limit of 9.2 × 103 particles mL-1 with a linear range of 4 × 104 to 5 × 107 particles mL-1 for uEVs. Moreover, the multimodal data fusion model exhibited an accuracy of 95.0%, a precision of 93.1%, and a recall rate of 93.2% on average, while those of the three types of unimodal models were no more than 91%. The elevated diagnosis precision by using the present fusion platform offers a perspective approach to diminishing the rate of misdiagnosis and overtreatment of BCa.

Manganese-enriched prussian blue nanohybrids with smaller electrode potential and lower charge transfer resistance to enhance combination therapy.

Prussian blue (PB) is authenticated in clinical treatment, while it generally exhibits unfavorable chemodynamic therapy (CDT) performance. Herein, we developed manganese-doped prussian blue (PBM) nanoparticles to significantly enhance both CDT and photothermal therapy (PTT) effect. The lower redox potential of Mn3+/2+ (0.088 V) in PBM against that of Fe2+/3+ (0.192 V) in PB leads to favorable electron transfer of PBM with respect to PB. Besides, PBM has a lower charge-transfer resistance (Rct) of 2.98 Ω than 4.83 Ω of PB. Once PBM entering the tumor microenvironment (TME), Mn3+ may be readily reduced by glutathione (GSH) and therein to enhance intracellular oxidative stress. Meanwhile, the superoxide dismutase (SOD)-like activity of PBM facilitates the conversion of endogenous superoxide (O2•-) into H2O2. Mn2+ subsequently catalyzes H2O2 to generate toxic hydroxyl radicals (•OH). Notably, the PBM plus laser irradiation can effectively trigger a robust immunogenic cell death (ICD) due to the combination therapy of CDT and PTT. Additionally, the mice treated by PBM followed by laser irradiation efficiently avoided splenomegaly and lung metastasis, along with significant up-regulation of the Stimulator of Interferon Genes (STING) expression. Overall, PBM significantly inhibits tumor growth and metastasis, making it a promising multifunctional nanoplatform for cancer treatment.

An extracellular vesicle microRNA-initiated 3D DNAzyme motor for colorectal cancer diagnosis.

MicroRNA is regarded as a significant biomarker for cancer diagnosis, disease process evaluation and therapeutic guidance, and dual-parameter measurement may contribute to a more accurate and realistic assessment. To meet the urgent need for simultaneous detection of multiple biomarkers, we combined three-dimensional DNAzyme motors with single molecule imaging technique to construct a convenient, intuitive, and sensitive approach for the simultaneous detection of dual miRNAs in the free state or in extracellular vesicles. Quantification of target miRNAs can be realized through the detection of amplified fluorescence signals generated by the target miRNA-initiated cleavage of fluorescent substrate strands by the DNAzyme motors. The practicability was systematically validated with microRNA-21-5p and microRNA-10b-5p as targets, acquiring a satisfactory sensitivity sufficient to detect low abundance targets at 0.5 or 1 pM to 100 pM. Besides, the extracellular vesicular miRNAs can be conveniently detected without extraction. The clinical applicability was verified with a series of extracellular vesicles from clinical samples, which exhibited good distinguishability between colorectal cancer patients and healthy donors. In addition to the advantages of good specificity and high sensitivity, the system has potential to be easily adapted by minor alteration of the DNA sequences and fluorophore sets for detection of multiple miRNAs and even other types of biomarkers such as proteins. Therefore, it shows promise to be widely applied in various fields such as early diagnosis of cancer and its prognostic assessment.

Pro-CRISPR PcrIIC1-associated Cas9 system for enhanced bacterial immunity.

The CRISPR system is an adaptive immune system found in prokaryotes that defends host cells against the invasion of foreign DNA1. As part of the ongoing struggle between phages and the bacterial immune system, the CRISPR system has evolved into various types, each with distinct functionalities2. Type II Cas9 is the most extensively studied of these systems and has diverse subtypes. It remains uncertain whether members of this family can evolve additional mechanisms to counter viral invasions3,4. Here we identify 2,062 complete Cas9 loci, predict the structures of their associated proteins and reveal three structural growth trajectories for type II-C Cas9. We found that novel associated genes (NAGs) tended to be present within the loci of larger II-C Cas9s. Further investigation revealed that CbCas9 from Chryseobacterium species contains a novel ß-REC2 domain, and forms a heterotetrameric complex with an NAG-encoded CRISPR-Cas-system-promoting (pro-CRISPR) protein of II-C Cas9 (PcrIIC1). The CbCas9-PcrIIC1 complex exhibits enhanced DNA binding and cleavage activity, broader compatibility for protospacer adjacent motif sequences, increased tolerance for mismatches and improved anti-phage immunity, compared with stand-alone CbCas9. Overall, our work sheds light on the diversity and 'growth evolutionary' trajectories of II-C Cas9 proteins at the structural level, and identifies many NAGs-such as PcrIIC1, which serves as a pro-CRISPR factor to enhance CRISPR-mediated immunity.

Dynamic Stomach Model-Capillary Electrophoresis-ICPMS for Evaluation of Release and Transformation Behaviors of Arsenic Species from Microplastics during Digestion.

Microplastics (MPs) can act as carriers of environmental arsenic species into the stomach with food and release arsenic species during digestion, which threatens human health. Herein, an integrated dynamic stomach model (DSM)-capillary electrophoresis-inductively coupled plasma mass spectrometry (CE-ICPMS) is developed for online monitoring of the release and transformation behaviors of arsenic species loaded on MPs (As-MPs) in the simulated human stomach. The 3D-printed DSM with a soft stomach chamber enables the behaviors of gastric peristalsis, gastric and salivary fluid addition, pH adjustment, and gastric emptying (GE) to be controlled by a self-written program after oral ingestion of food with As-MPs. The gastric extract during digestion is introduced into the spiral channel to remove the large particulate impurity and online filtered to obtain the clarified arsenic-containing solution for subsequent speciation analysis of arsenic by CE-ICPMS. The digestion conditions and pretreatment processes of DSM are tracked and validated, and the release rates of As-MPs digested by DSM are compared with those digested by the static stomach model and DSM without GE. The release rate of inorganic arsenic on MPs is higher than that of organic arsenic throughout the gastric digestion process, and 8% of As(V) is reduced to As(III). The detection limits for As(III), DMA, MMA, and As(V) are 0.5-0.9 µg L-1 using DSM-CE-ICPMS, along with precisions of ≤8%. This present method provides an integrated and convenient tool for evaluating the release and transformation of As-MPs during human gastric digestion and provides a reference for exploring the interactions between MPs and metals/metalloids in the human body.

Mast cell activation triggered by SARS-CoV-2 causes inflammation in brain microvascular endothelial cells and microglia.

SARS-CoV-2-induced excessive inflammation in brain leads to damage of blood-brain barrier, hypoxic-ischemic injury, and neuron degeneration. The production of inflammatory cytokines by brain microvascular endothelial cells and microglia is reported to be critically associated with the brain pathology of COVID-19 patients. However, the cellular mechanisms for SARS-CoV-2-inducing activation of brain cells and the subsequent neuroinflammation remain to be fully delineated. Our research, along with others', has recently demonstrated that SARS-CoV-2-induced accumulation and activation of mast cells (MCs) in mouse lung could further induce inflammatory cytokines and consequent lung damages. Intracerebral MCs activation and their cross talk with other brain cells could induce neuroinflammation that play important roles in neurodegenerative diseases including virus-induced neuro-pathophysiology. In this study, we investigated the role of MC activation in SARS-CoV-2-induced neuroinflammation. We found that (1) SARS-CoV-2 infection triggered MC accumulation in the cerebrovascular region of mice; (2) spike/RBD (receptor-binding domain) protein-triggered MC activation induced inflammatory factors in human brain microvascular endothelial cells and microglia; (3) MC activation and degranulation destroyed the tight junction proteins in brain microvascular endothelial cells and induced the activation and proliferation of microglia. These findings reveal a cellular mechanism of SARS-CoV-2-induced neuroinflammation.

Deep Learning Promotes Profiling of Multiple miRNAs in Single Extracellular Vesicles for Cancer Diagnosis.

Extracellular vesicle microRNAs (EV miRNAs) are critical noninvasive biomarkers for early cancer diagnosis. However, accurate cancer diagnosis based on bulk analysis is hindered by the heterogeneity among EVs. Herein, we report an approach for profiling single-EV multi-miRNA signatures by combining total internal reflection fluorescence (TIRF) imaging with a deep learning (DL) algorithm for the first time. This innovative technique allows for the precise characterization of EV miRNAs at the single-vesicle level, overcoming the challenges posed by EV heterogeneity. TIRF with high resolution and a signal-to-noise ratio can simultaneously detect multi-miRNAs in situ in individual EVs. DL algorithm avoids complicated and inaccurate artificial feature extraction, achieving automated high-resolution image analysis. Using this approach, we reveal that the main variation of EVs from 5 cancer cells and normal plasma is the triple-positive EV subpopulation, and the classification accuracy of single triple-positive EVs from 6 sources can reach above 95%. In the clinical cohort, 20 patients (5 lung cancer, 5 breast cancer, 5 cervical cancer, and 5 colon cancer) and 5 healthy controls are predicted with an overall accuracy of 100%. This single-EV strategy provides new opportunities for exploring more specific EV biomarkers to achieve cancer diagnosis and classification.

A hydrodynamic-based dual-function microfluidic chip for high throughput discriminating tumor cells.

A hydrodynamic-based microfluidic chip consisted of two function units that could not only separate tumor cells (TCs) from whole blood but also remove residual blood cells was designed. The separation of TCs was achieved by a straight contraction-expansion array (CEA) microchannel on the front end of the chip. The addition of contractive structure brought a micro-vortex like Dean vortex that promoted cell focusing in the channel, while when cells entered the dilated region, the wall-induced lift force generated by the channel wall gave cells a push away from the wall. As the wall-induced lift force is proportional to the third power of the cell diameter, TCs with larger diameter will have a larger lateral migration under the wall-induced lift force, realizing the separation of TCs from blood sample. Fluorescent particles with diameters of 19.3 µm and 4.5 µm were used to simulate TCs and red blood cells, respectively, to verify the separation capacity of the proposed CEA microchannel for particles with different diameter. And a separation efficiency 98.7% for 19.3 µm particles and a removal rate 96.2% for 4.5 µm particles was observed at sample flow rate of 10 µL min-1 and sheath flow rate of 190 µL min-1. In addition, a separation efficiency about 96.1% for MCF-7 cells (stained with DiI) and removal rates of 96.2% for red blood cells (RBCs) and 98.7% for white blood cells (WBCs) were also obtained under the same condition. However, on account of the large number of blood cells in the blood, there will be a large number of blood cells remained in the isolated TCs, so a purification unit based on hydrodynamic filtration (HDF) was added after the separation microchannel. The purification channel is a size-dictated cell filter that can remove residual blood cells but retain TCs, thus achieving the purification of TCs. Combined the CEA microchannel and the purifier, the microchip facilitates sorting of MCF-7 cells from whole blood with a separation rate about 95.3% and a removal rate over 99.99% for blood cells at a sample flow rate of 10 µL min-1, sheath flow rate of 190 µL min-1 and washing flow rate of 63 µL min-1.

Mast cell degranulation-triggered by SARS-CoV-2 induces tracheal-bronchial epithelial inflammation and injury.

SARS-CoV-2 infection-induced hyper-inflammation is a key pathogenic factor of COVID-19. Our research, along with others', has demonstrated that mast cells (MCs) play a vital role in the initiation of hyper-inflammation caused by SARS-CoV-2. In previous study, we observed that SARS-CoV-2 infection induced the accumulation of MCs in the peri-bronchus and bronchioalveolar-duct junction in humanized mice. Additionally, we found that MC degranulation triggered by the spike protein resulted in inflammation in alveolar epithelial cells and capillary endothelial cells, leading to subsequent lung injury. The trachea and bronchus are the routes for SARS-CoV-2 transmission after virus inhalation, and inflammation in these regions could promote viral spread. MCs are widely distributed throughout the respiratory tract. Thus, in this study, we investigated the role of MCs and their degranulation in the development of inflammation in tracheal-bronchial epithelium. Histological analyses showed the accumulation and degranulation of MCs in the peri-trachea of humanized mice infected with SARS-CoV-2. MC degranulation caused lesions in trachea, and the formation of papillary hyperplasia was observed. Through transcriptome analysis in bronchial epithelial cells, we found that MC degranulation significantly altered multiple cellular signaling, particularly, leading to upregulated immune responses and inflammation. The administration of ebastine or loratadine effectively suppressed the induction of inflammatory factors in bronchial epithelial cells and alleviated tracheal injury in mice. Taken together, our findings confirm the essential role of MC degranulation in SARS-CoV-2-induced hyper-inflammation and the subsequent tissue lesions. Furthermore, our results support the use of ebastine or loratadine to inhibit SARS-CoV-2-triggered degranulation, thereby preventing tissue damage caused by hyper-inflammation.