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.

An EMC-Hpo-Yki axis maintains intestinal homeostasis under physiological and pathological conditions.

Balanced control of stem cell proliferation and differentiation underlines tissue homeostasis. Disruption of tissue homeostasis often results in many diseases. However, how endogenous factors influence the proliferation and differentiation of intestinal stem cells (ISCs) under physiological and pathological conditions remains poorly understood. Here, we find that the evolutionarily conserved endoplasmic reticulum membrane protein complex (EMC) negatively regulates ISC proliferation and intestinal homeostasis. Compromising EMC function in progenitors leads to excessive ISC proliferation and intestinal homeostasis disruption. Mechanistically, the EMC associates with and stabilizes Hippo (Hpo) protein, the key component of the Hpo signaling pathway. In the absence of EMC, Yorkie (Yki) is activated to promote ISC proliferation due to Hpo destruction. The EMC-Hpo-Yki axis also functions in enterocytes to maintain intestinal homeostasis. Importantly, the levels of the EMC are dramatically diminished in tunicamycin-treated animals, leading to Hpo destruction, thereby resulting in intestinal homeostasis disruption due to Yki activation. Thus, our study uncovers the molecular mechanism underlying the action of the EMC in intestinal homeostasis maintenance under physiological and pathological conditions and provides new insight into the pathogenesis of tunicamycin-induced tumorigenesis.

Diamond controls epithelial polarity through the dynactin-dynein complex.

Epithelial polarity is critical for proper functions of epithelial tissues, tumorigenesis, and metastasis. The evolutionarily conserved transmembrane protein Crumbs (Crb) is a key regulator of epithelial polarity. Both Crb protein and its transcripts are apically localized in epithelial cells. However, it remains not fully understood how they are targeted to the apical domain. Here, using Drosophila ovarian follicular epithelia as a model, we show that epithelial polarity is lost and Crb protein is absent in the apical domain in follicular cells (FCs) in the absence of Diamond (Dind). Interestingly, Dind is found to associate with different components of the dynactin-dynein complex through co-IP-MS analysis. Dind stabilizes dynactin and depletion of dynactin results in almost identical defects as those observed in dind-defective FCs. Finally, both Dind and dynactin are also required for the apical localization of crb transcripts in FCs. Thus our data illustrate that Dind functions through dynactin/dynein-mediated transport of both Crb protein and its transcripts to the apical domain to control epithelial apico-basal (A/B) polarity.

The Yun/Prohibitin complex regulates adult Drosophila intestinal stem cell proliferation through the transcription factor E2F1.

Stem cells constantly divide and differentiate to maintain adult tissue homeostasis, and uncontrolled stem cell proliferation leads to severe diseases such as cancer. How stem cell proliferation is precisely controlled remains poorly understood. Here, from an RNA interference (RNAi) screen in adult Drosophila intestinal stem cells (ISCs), we identify a factor, Yun, required for proliferation of normal and transformed ISCs. Yun is mainly expressed in progenitors; our genetic and biochemical evidence suggest that it acts as a scaffold to stabilize the Prohibitin (PHB) complex previously implicated in various cellular and developmental processes and diseases. We demonstrate that the Yun/PHB complex is regulated by and acts downstream of EGFR/MAPK signaling. Importantly, the Yun/PHB complex interacts with and positively affects the levels of the transcription factor E2F1 to regulate ISC proliferation. In addition, we find that the role of the PHB complex in cell proliferation is evolutionarily conserved. Thus, our study uncovers a Yun/PHB-E2F1 regulatory axis in stem cell proliferation.

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.

Urine Self-Sampling Kit Combined with an Automated Preparation-Sampler Device for Convenient and Reliable Analysis of Arsenic Metabolites by HPLC-ICPMS.

Speciation analysis of arsenic in urine is essential for the studies of arsenic metabolism and biological effects, but the unstable arsenic species represented by MMAIII and DMAIII pose a huge challenge to analytical accuracy. Herein, a novel urine self-sampling (USS) kit combined with an automated preparation-sampler (APS) device is rationally designed and used for convenient analysis of arsenic metabolites by high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICPMS). The subject can collect urine into a sampling vial at home and use a homemade syringe to pump argon to displace oxygen in the vial, thereby inhibiting the oxidation of MMAIII and DMAIII. After USS and transportation, the sampling vial is loaded directly onto the APS device, where the urine sample can be automatically mixed with diluent, filtered, and loaded into HPLC-ICPMS for arsenic speciation analysis under anaerobic conditions. For a single sample, the sampling time and the analysis time are <8 and <18 min, respectively. The recoveries of MMAIII and DMAIII in urine over 24 h at 4 °C are 86 and 67%, surpassing the conventional sampling method by 28 and 67%, respectively. When the APS is coupled to HPLC-ICPMS, the detection limits of AsC, iAsIII, MMAIII, DMAV, MMAV, DMAIII, and iAsV are 0.03-0.10 µg L-1 with precisions of <10%. The present method provides a convenient and reliable tool for the storage and analysis of unstable arsenic species in urine and lays the foundation for studying the metabolic and biological effects of methylated trivalent arsenicals.

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.

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.

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.

A Portable Microplasma Optical Emission Spectrometric Device with Online Digestion Function for Field and Sensitive Determination of Lead in Biological Samples.

Accurate detection and screening of Pb in biological samples is helpful to assess the risk associated with lead pollution to human health. However, conventional atomic spectroscopic instruments are bulky and cumbersome, requiring additional sample pretreatment equipment, and difficult to perform field analysis with. Herein, a portable point discharge (PD) microplasma-optical emission spectrometric (OES) device with online digestion function is designed for field and sensitive determination of lead in biological samples. With rice as a model, online digestion of a batch of six 50 mg samples can be achieved in the HNO3 and H2O2 system within 25 min by a temperature control and timing module. Compared to the conventional microwave digestion, the digestion efficiency of this device reaches 97%. Pb in digestion solution is converted into volatile species by hydride generation (HG) and directly introduced into PD-OES for excitation and detection by a self-designed rotatable and telescopic cutoff gas sampling column. Six samples can be successively detected in 2 min, and argon consumption of the whole process is only <800 mL. Under the optimized conditions, the detection limit of Pb is 0.018 mg kg-1 (0.9 µg L-1) and precision is 3.6%. The accuracy and practicability of the present device are verified by measuring several certified reference materials and real biological samples. By virtue of small size (23.5 × 17 × 8.5 cm3), lightweight (2.5 kg), and low energy consumption (24.3 W), the present device provides a convenient tool for field analysis of toxic elements in biological samples.

Hirudin ameliorates myocardial ischemia-reperfusion injury in a rat model of hemorrhagic shock and resuscitation: roles of NLRP3-signaling pathway.

Severe hemorrhage shock and resuscitation (HSR) has been reported to induce myocardial ischemia-reperfusion injury (MIRI), resulting in a poor prognosis. Hirudin, an effective thrombin inhibitor, can offer protection against MIRI. This study aimed to determine if hirudin administration ameliorates HSR-induced MIRI and the underlying mechanism. A rat model of HSR was established by bleeding rats to a mean arterial blood pressure of 30-35 mmHg for 45 min and then resuscitating them with all the shed blood through the left femoral vein. After HSR, 1 mg/kg of hirudin was administrated immediately. At 24 h after HSR, the cardiac injury was assessed using serum CK-MB, cTnT, hematoxylin-eosin (HE) staining, echocardiography, M1-polarized macrophages, and pyroptosis-associated factors, including cleaved caspase-1, Gasdermin D (GSDMD) N-terminal, IL-1ß, and IL-18 were measured by immunofluorescence and western blot assays. Nigericin, a unique agonist, was utilized to evaluate the responsibilities of NLRP3 signaling. Under the HSR condition, rats exhibited a significant increase in myocardial injury score, an elevation of serum cTnT, CK-MB levels, an aggrandization of M1-polarized macrophages, an upregulation of pyroptosis-associated factors, including cleaved caspase-1, GSDMD N-terminal, IL-1ß, and IL-18, but a significant decrease in left ventricular ejection fraction (EF%) and a reduction of left ventricular fractional shortening (FS%), while hirudin administration partially restored the changes. However, the NLRP3 agonist nigericin reversed the cardioprotective effects of hirudin. We determined the cardioprotective effects of hirudin against HSR-induced MIRI. The mechanism may involve the inhibition of NLRP3-induced pyroptosis.

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.

Occurrence of Chlorinated Derivatives of Bisphenol S in Paper Products and Their Potential Health Risks through Dermal Exposure.

The occurrence of chlorinated derivatives of bisphenol S (Clx-BPS) and BPS was investigated in nine types of paper products (n = 125), including thermal paper, corrugated boxes, mail envelopes, newspapers, flyers, magazines, food contact paper, household paper, and business cards. BPS was found in all paper product samples, while Clx-BPS were mainly found in thermal paper (from below the limit of detection (

Simultaneous Imaging of Multiple miRNAs in Mitochondria Controlled by Fluorescently Encoded Upconversion Optical Switches for Drug Resistance Studies.

Mitochondrial miRNAs (mitomiRs) are essential regulators of biological processes by influencing mitochondrial gene expression and function. To comprehensively understand related pathological processes and treatments, simultaneous imaging of multiple mitomiRs is crucial. In this study, we present a technique that enables simultaneous monitoring of multiple mitomiRs in living cells using a near-infrared (NIR) photoactivated controlled detection probe (PD-mFleU) with a fluorescence-encoded error correction module and a nonsupervised machine learning data-processing algorithm. This method allows controlled sensing imaging of mitomiRs with a DNA reporter probe that can be activated by NIR light after targeted mitochondrial localization. Multilayer upconversion nanoparticles (UCNPs) are used for encoding probes and error correction. Additionally, the density-based spatial clustering of applications with the noise (DBSCAN) algorithm is used to process and analyze the image. Using this technique, we achieved rapid in situ imaging of the abnormal expression of three mitomiRs (miR-149, miR-590, and miR-671) related to mt-ND1 in drug-resistant cells. Furthermore, upregulating the three mitomiRs simultaneously efficiently reverted drug-resistant cells to sensitive cells. Our study provides an analytical strategy for multiplex imaging of mitomiRs in living cells with potential clinical applications.

One-Pot Pretreatment Coupled to Microplasma Optical Emission Spectrometry for Field and Sensitive Determination of Inorganic Mercury and Methylmercury in Fish.

Field and sensitive analysis of mercury species in seafood is helpful to assess the risk of human exposure to mercury, but the cumbersome pretreatment process is time-consuming and laborious. Herein, a simple one-pot pretreatment system is designed for extraction, separation, and enrichment of inorganic mercury (Hg(II)) and methylmercury (MeHg) in fish, and coupled to dielectric barrier discharge (DBD) microplasma optical emission spectrometry (OES). Both Hg(II) and MeHg species in fish can be effectively extracted by tetramethylammonium hydroxide under ultrasound, then separated from the fish matrix by vapor generation and photochemical vapor generation, and finally enriched on the activated carbon electrode tips. Mercury trapped on the activated carbon electrode tips can be rapidly released to produce OES under the DBD microplasma excitation for quantitative analysis. The pretreatment and analysis of a batch of 12 samples are completed within 50 min, and the extraction efficiency of total mercury is up to 90% for 100 mg of freeze-dried fish or 86% for 1 g of fresh fish. Under the optimized conditions, the detection limits are 2 µg kg-1 for Hg(II) and 1.2 µg kg-1 for MeHg in freeze-dried fish, and precisions are 3.2% for Hg(II) and 3.9% for MeHg. The present method is applied to the analysis of the certified reference material and real marine fishes, giving rise to spiked recoveries of 95-103%. The present system hardly leads to MeHg and Hg(II) transforming into each other during extraction, providing a simple, convenient, and low-cost analytical tool to evaluate the risk of mercury species in fish.

Facile Lego-Spinner Pretreatment Device for Analysis of Arsenic Species in Dried Blood Spots by Ion Chromatography-Inductively Coupled Plasma-Mass Spectrometry.

Dried blood spot (DBS) detection has the advantages of small blood collection, convenience, and reliability, which provides a possibility for large-scale evaluation of arsenic exposure in human population. Herein, a facile Lego-spinner pretreatment device is rationally designed for speciation analysis of arsenic in DBSs by ion chromatography-inductively coupled plasma-mass spectrometry (IC-ICP-MS). In the mixing mode of the Lego-spinner, the magnetic stir bar in the centrifuge tube rotates under a magnetic field to assist the dispersive extraction of arsenic species in the DBS with reagents. In the centrifugation mode of the Lego-spinner, the arsenic extract is separated from the blood matrix for the subsequent IC-ICP-MS analysis. For the DBS prepared from 80 µL of whole blood, the whole pretreatment operation can be completed within 25 min. The detection limits of arsenobetaine, arsenite, dimethylarsenate, monomethylarsonate, and arsenate in the DBS are 0.09-0.15 µg L-1, and precisions are <11%. The concentrations of these five arsenic species are highly correlated between whole blood and the DBS (r2 > 0.97), and Bland-Altman analysis indicates that the concentration difference of arsenic species between whole blood and the DBS is within ±20%. The DBS sampling approach can effectively preserve arsenic species for at least 30 days at 4 °C, and the contents of arsenic species in the DBS prepared from capillary blood are in a reasonable agreement with those of venous whole blood (gold standard). This Lego-spinner provides a handy and efficient tool for fast extraction of arsenic species in DBSs, facilitating the in-depth study of arsenic migration and transformation in the human body.

Two-Dimensional Multi-parameter Cytometry Platform for Single-Cell Analysis.

A 2D flow cytometry platform, known as CytoLM Plus, was developed for multi-parameter single-cell analysis. Single particles or cells after hydrodynamic alignment in a microfluidic unit undergo first-dimension fluorescence and side scattering dual-channel optical detection. They were thereafter immediately directed to ICP-MS by connecting the microfluidic unit with a high-efficiency nebulizer to facilitate the second-dimension ICP-MS detection. Flow cytometry measurements of fluorescent microspheres evaluated the performance of CytoLM Plus for optical detection. 6434 fluorescence bursts were observed with a valid signal proportion as high as 99.7%. After signal unification and gating analysis, 6067 sets of single-particle signals were obtained with 6.6 and 6.2% deviations for fluorescence burst area and height, respectively. This is fairly comparable with that achieved by a commercial flow cytometer. Afterward, CytoLM Plus was evaluated by 2D flow cytometry measurement of Ag+-incubated and AO-stained MCF-7 cells. A program for 2D single-cell signal unification was developed based on the algorithm of screening in lag time window. In the present case, a lag time window of -4.2 ± 0.09 s was determined by cross-correlation analysis and two-parameter optimization, which efficiently unified the concurrent single-cell signals from fluorescence, side scattering, and ICP-MS. A total of 495 sets of concurrent 2D signals were screened out, and the statistical analysis of these single-cell signals ensured 2D multi-parameter single-cell analysis and data elucidation.

Dual Functional Full-Color Carbon Dot-Based Organelle Biosensor Array for Visualization of Lipid Droplet Subgroups with Varying Lipid Composition in Living Cells.

In situ visualization of lipid composition diversity in lipid droplets (LDs) is essential for decoding lipid metabolism and function. However, effective probes for simultaneously localizing and reflecting the lipid composition of LDs are currently lacking. Here, we synthesized full-color bifunctional carbon dots (CDs) that can target LDs as well as respond to the nuance in internal lipid compositions with highly sensitive fluorescence signals, due to lipophilicity and surface state luminescence. Combined with microscopic imaging, uniform manifold approximation and projection, and sensor array concept, the capacity of cells to produce and maintain LD subgroups with varying lipid composition was clarified. Moreover, in oxidative stress cells, LDs with characteristic lipid compositions were deployed around mitochondria, and the proportion of LD subgroups changed, which gradually disappeared when treated with oxidative stress therapeutics. The CDs demonstrate great potential for in situ investigation of the LD subgroups and metabolic regulations.

Dean-Flow-Coupled Elasto-Inertial Focusing Accelerates Exosome Purification to Facilitate Single Vesicle Profiling.

Exosomes are recognized as noteworthy biomarkers playing unprecedented roles in intercellular communication and disease diagnosis and treatment. It is a prerequisite to obtain high-purity exosomes for the comprehension of exosome biochemistry and further illustration of their functionality/mechanisms. However, the isolation of nanoscale exosomes from endogenous proteins is particularly challenging for small-volume biological samples. Herein, a Dean-flow-coupled elasto-inertial microfluidic chip (DEIC) was developed. It consists of a spiral microchannel with dimensional confined concave structures and facilitates elasto-inertial separation of exosomes with lower protein contaminants from cell culture medium and human serum. The presence of 0.15% (w/v) poly-(oxyethylene) controls the elastic lift force acting on suspended nanoscale particles and makes it feasible for field-free purification of integrity exosomes with a 70.6% recovery and a 91.4% removal rate for proteins. As a proof of concept, the technique demonstrated the individual-vesicle-level biomarker (EpCAM and PD-L1) profiling in combination with simultaneous aptamer-mediated analysis to disclose the sensibility for immune response. Overall, DEIC enables the collection of high-purity exosomes and exhibits potential in integration with downstream analyses of exosomes.

Interaction of Cellular Uptake of Nanosilver and Metallothionein Stress Expression Elucidated by 2D Single-Cell Analyses Based on LIF and ICP-MS.

The exploration of cytology mechanisms of nanosilver uptake, toxicity, and detoxification has become an important issue due to its widespread applications. Previous studies have shown differences in the toxic response of mammalian cells to nanosilver. However, the analysis results based on cell populations ignore the impact of cell uptake heterogeneity on the expression of associated stress proteins and cellular physiological activities. In this respect, this work investigated the interaction between silver uptake and metallothionein (MT) expression in individual cells. In addition, we have also preliminarily elucidated the sensitivity variation to AgNPs by using five cell lines, e.g., LX-2, HepG-2, SK-HEP-1, Huh-7, and MDA-MB-231, by adopting a two-dimensional (2D) high-throughput single-cell analysis platform coupling laser-induced fluorescence (LIF) and inductively coupled plasma mass spectrometry (ICP-MS). We developed a 2D data analysis method for one-to-one unification of fluorescence-mass spectrometry signals corresponding to a specific single cell. It indicated that there is no obvious correlation between cellular silver uptake and cell size, and the low MT expression of cells is more sensitive to silver nanoparticles. For each cell line, significant heterogeneity in MT expression was observed. This provides important information for understanding the potential heterogeneous effects of nanosilver on mammalian biological systems. Overall, detoxified cells are more tolerant to nanosilver and normal cells are more tolerant than cancer cells.