Association between the dietary antioxidant index and relative telomere length of leucocytes in the Chinese population.

Dietary antioxidant indices (DAI) may be potentially associated with relative telomere length (RTL) of leucocytes. This study aimed to investigate the relationship between DAI and RTL. A cross-sectional study involving 1656 participants was conducted. A generalised linear regression model and a restricted cubic spline model were used to assess the correlation of DAI and its components with RTL. Generalised linear regression analysis revealed that DAI (ß = 0·005, P = 0·002) and the intake of its constituents vitamin C (ß = 0·043, P = 0·027), vitamin E (ß = 0·088, P < 0·001), Se (ß = 0·075, P = 0·003), and Zn (ß = 0·075, P = 0·023) were significantly and positively correlated with RTL. Sex-stratified analysis showed that DAI (ß = 0·006, P = 0·005) and its constituents vitamin E (ß = 0·083, P = 0·012), Se (ß = 0·093, P = 0·006), and Zn (ß = 0·092, P = 0·034) were significantly and positively correlated with RTL among females. Meanwhile, among males, only vitamin E intake (ß = 0·089, P = 0·013) was significantly and positively associated with RTL. Restricted cubic spline analysis revealed linear positive associations between DAI and its constituents' (vitamin E, Se and Zn) intake and RTL in the total population. Sex-stratified analysis revealed a linear positive correlation between DAI and its constituents' (vitamin E, Se and Zn) intake and RTL in females. Our study found a significant positive correlation between DAI and RTL, with sex differences.

Association between multiple-heavy-metal exposures and systemic immune inflammation in a middle-aged and elderly Chinese general population.

BACKGROUND: Exposure to heavy metals alone or in combination can promote systemic inflammation. The aim of this study was to investigate potential associations between multiple plasma heavy metals and markers of systemic immune inflammation. METHODS: Using a cross-sectional study, routine blood tests were performed on 3355 participants in Guangxi, China. Eight heavy metal elements in plasma were determined by inductively coupled plasma mass spectrometry. Immunoinflammatory markers were calculated based on peripheral blood WBC and its subtype counts. A generalised linear regression model was used to analyse the association of each metal with the immunoinflammatory markers, and the association of the metal mixtures with the immunoinflammatory markers was further assessed using weighted quantile sum (WQS) regression. RESULTS: In the single-metal model, plasma metal Fe (log10) was significantly negatively correlated with the levels of immune-inflammatory markers SII, NLR and PLR, and plasma metal Cu (log10) was significantly positively correlated with the levels of immune-inflammatory markers SII and PLR. In addition, plasma metal Mn (log10 conversion) was positively correlated with the levels of immune inflammatory markers NLR and PLR. The above associations remained after multiple corrections. In the mixed-metal model, after WQS regression analysis, plasma metal Cu was found to have the greatest weight in the positive effects of metal mixtures on SII and PLR, while plasma metals Mn and Fe had the greatest weight in the positive effects of metal mixtures on NLR and LMR, respectively. In addition, blood Fe had the greatest weight in the negative effects of the metal mixtures for SII, PLR and NLR. CONCLUSION: Plasma metals Cu and Mn were positively correlated with immunoinflammatory markers SII, NLR and PLR. While plasma metal Fe was negatively correlated with immunoinflammatory markers SII, NLR, and PLR.

Microfluidic Cell Trap Arrays for Single Hematopoietic Stem/Progenitor Cell Behavior Analysis.

Hematopoietic stem/progenitor cell (HSPC) mobilization from the bone marrow to the bloodstream is a required step for blood cell renewal, and HSPC motility is a clinically relevant standard for peripheral blood stem cell transplantation. Individual HSPCs exhibit considerable heterogeneity in motility behaviors, which are subject to complex intrinsic and extrinsic regulatory mechanisms. Motility-based cell sorting is then demanded to fulfill the study of such mechanism complexity. However, due to the HSPC heterogeneity and difficulty in monitoring cell motility, such a platform is still not available. With the recent development of microfluidics technology, motility-based monitoring, sorting, collecting, and analysis of HSPC behaviors are highly possible and achievable if fluid channels and structures are correctly engineered. Here, a new design of microfluidic arrays for single-cell trapping is presented, enabling high-throughput analysis of individual HSPC motility and behavior. Using these arrays, it is observed that HSPC motility is positively correlated with CD34 asymmetric inheritance and cell differentiation. Transcriptomic analysis of HSPCs sorted according to motility reveals changes in expression of genes associated with the regulation of stem-cell maintenance. Ultimately, this novel, physical cell-sorting system can facilitate the screening of HSPC mobilization compounds and the analysis of signals driving HSPC fate decisions.

Microfluidics-Based Single-Cell Protrusion Analysis for Screening Drugs Targeting Subcellular Mitochondrial Trafficking in Cancer Progression.

Cancer cell migration is often guided by cell protrusions, whose formation and activity involve subcellular localization of mitochondria. However, the role of subcellular mitochondrial trafficking during cell protrusion generation is not well-understood amidst a lack of quantitative data. Here, we present a high-throughput microfluidic platform that enables the quantitative, single-cell precision analysis of cell protrusion formation during cell migration that is regulated by subcellular mitochondrial trafficking. Gene expression profiling of the isolated cell protrusions suggested that mitochondria were found in high numbers within cell protrusions, a finding validated by mitochondrial staining. Quantitative analysis revealed that the formation of cell protrusions could be effectively suppressed by inhibiting subcellular mitochondrial trafficking. We further demonstrated that rapid screening of mitochondria-specific therapeutic drugs to evaluate their effects on cell protrusion formation with single-cell precision could be achieved in the microfluidic platform, which could have clinical utility in the development of new anticancer agents.

CRISPR-Cas12a Coupled with Platinum Nanoreporter for Visual Quantification of SNVs on a Volumetric Bar-Chart Chip.

Methods that can detect and quantify single nucleotide variations (SNVs)/single nucleotide polymorphisms (SNPs) are greatly needed in the bioanalytical measurement of gene mutations and polymorphisms. Herein a visual and instrument-free SNV quantification platform is developed. Platinum nanoparticles tethered to magnetic beads by single-stranded DNAs are designed as quantitative readout reporters for a CRISPR-Cas12a nucleic acid detection system. The integration of platinum nanoreporter and CRISPR-Cas system with a volumetric bar-chart chip realizes the volumetric quantification of nucleic acids. This platform enables quantification of multiple cancer mutations in pure DNA samples and mock cell-free DNA samples in serum, with allelic fractions as low as 0.01%. This platform could have great potential in the quantification of SNVs/SNPs as well as other types of nucleic acid targets at the point of care.

Immunological Synapse Predicts Effectiveness of Chimeric Antigen Receptor Cells.

Chimeric antigen receptor (CAR)-modified T cell therapy has the potential to improve the overall survival of patients with malignancies by enhancing the effectiveness of CAR T cells. Precisely predicting the effectiveness of various CAR T cells represents one of today's key unsolved problems in immunotherapy. Here, we predict the effectiveness of CAR-modified cells by evaluating the quality of the CAR-mediated immunological synapse (IS) by quantitation of F-actin, clustering of tumor antigen, polarization of lytic granules (LGs), and distribution of key signaling molecules within the IS. Long-term killing capability, but not secretion of conventional cytokines or standard 4-hr cytotoxicity, correlates positively with the quality of the IS in two different CAR T cells that share identical antigen specificity. Xenograft model data confirm that the quality of the IS in vitro correlates positively with performance of CAR-modified immune cells in vivo. Therefore, we propose that the quality of the IS predicts the effectiveness of CAR-modified immune cells, which provides a novel strategy to guide CAR therapy.

PD-1 blocks lytic granule polarization with concomitant impairment of integrin outside-in signaling in the natural killer cell immunological synapse.

BACKGROUND: The inhibitory receptor programmed cell death protein 1 (PD-1) is upregulated on a variety of immune cells, including natural killer (NK) cells, during chronic viral infection and tumorigenesis. Blockade of PD-1 or its ligands produces durable clinical responses with tolerable side effects in patients with a broad spectrum of cancers. However, the underlying molecular mechanisms of how PD-1 regulates NK cell function remain poorly characterized. OBJECTIVE: We sought to determine the effect of PD-1 signaling on NK cells. METHODS: PD-1 was overexpressed in CD16-KHYG-1 (a human NK cell line with both antibody-dependent cellular cytotoxicity through CD16 and natural cytotoxicity through NKG2D) cells and stimulated by exposing the cells to NK-sensitive target cells expressing programmed death ligand 1 (PD-L1). RESULTS: PD-1 engagement by PD-L1 specifically blocked NK cell-mediated cytotoxicity without interfering with the conjugation between NK cells and target cells. Further examination showed that PD-1 signaling blocked lytic granule polarization in NK cells, which was accompanied by failure of integrin-linked kinase, a key molecule in the integrin outside-in signaling pathway, to accumulate in the immunological synapse after NK-target cell conjugation. CONCLUSION: Our results suggest that NK cell cytotoxicity is inhibited by PD-1 engagement, which blocks lytic granule polarization to the NK cell immunological synapse with concomitant impairment of integrin outside-in signaling. This study provides novel mechanistic insights into how PD-1 inhibition disrupts NK cell function.

High-Throughput Isolation of Cell Protrusions with Single-Cell Precision for Profiling Subcellular Gene Expression.

Invading cancer cells extend cell protrusions, which guide cancer-cell migration and invasion, eventually leading to metastasis. The formation and activity of cell protrusions involve the localization of molecules and organelles at the cell front; however, it is challenging to precisely isolate these subcellular structures at the single-cell level for molecular analysis. Here, we describe a newly developed microfluidic platform capable of high-throughput isolation of cell protrusions at single-cell precision for profiling subcellular gene expression. Using this microfluidic platform, we demonstrate the efficient generation of uniform cell-protrusion arrays (more than 5000 cells with protrusions) for a series of cell types. We show precise isolation of cell protrusions with high purity at single-cell precision for subsequent RNA-Seq analysis, which was further validated by RT-qPCR and RNA FISH. Our highly controlled protrusion isolation method opens a new avenue for the study of subcellular functional mechanisms and signaling pathways in metastasis.

Direct Assessment of the Toxicity of Molybdenum Disulfide Atomically Thin Film and Microparticles via Cytotoxicity and Patch Testing.

The low toxicity of molybdenum disulfide (MoS2 ) atomically thin film and microparticles is confirmed via cytotoxicity and patch testing in this report. The toxicity of MoS2 thin film and microparticles is extensively studied but is still inconclusive due to potential organic contamination in the preparations of samples. Such contamination is avoided here through preparing MoS2 atomically thin film via direct sulfurization of molybdenum thin film on quartz plate, which permits a direct assessment of its toxicity without any contamination. Six different types of cells, including normal, cancer, and immortal cells, are cultured in the media containing MoS2 thin film on quartz plates or dispersed MoS2 microparticles and their viability is evaluated with respect to the concentrations of samples. Detached thin films from the quartz plates are also investigated to estimate the toxicity of dispersed MoS2 in biological media. Allergy testing on skin of guinea pigs is also conducted to understand their effect on animal skins. By avoiding possible organic contamination, the low toxicity of MoS2 atomically thin film and microparticles to cells and animal skins paves the way for its applications in flexible biosensing/bioimaging devices and biocompatible coatings.

High-throughput analysis of yeast replicative aging using a microfluidic system.

Saccharomyces cerevisiae has been an important model for studying the molecular mechanisms of aging in eukaryotic cells. However, the laborious and low-throughput methods of current yeast replicative lifespan assays limit their usefulness as a broad genetic screening platform for research on aging. We address this limitation by developing an efficient, high-throughput microfluidic single-cell analysis chip in combination with high-resolution time-lapse microscopy. This innovative design enables, to our knowledge for the first time, the determination of the yeast replicative lifespan in a high-throughput manner. Morphological and phenotypical changes during aging can also be monitored automatically with a much higher throughput than previous microfluidic designs. We demonstrate highly efficient trapping and retention of mother cells, determination of the replicative lifespan, and tracking of yeast cells throughout their entire lifespan. Using the high-resolution and large-scale data generated from the high-throughput yeast aging analysis (HYAA) chips, we investigated particular longevity-related changes in cell morphology and characteristics, including critical cell size, terminal morphology, and protein subcellular localization. In addition, because of the significantly improved retention rate of yeast mother cell, the HYAA-Chip was capable of demonstrating replicative lifespan extension by calorie restriction.

Fast, Sensitive, and Quantitative Point-of-Care Platform for the Assessment of Drugs of Abuse in Urine, Serum, and Whole Blood.

Drug abuse is a major public health problem in many countries in Europe and North America. Currently available platforms for drug abuse assessment are facing technical challenges of nonquantitation, inaccuracy, low throughput, incompatibility with diverse complex specimens, long assay times, and requirement of instrument and/or expertise for readout. Here, we report an integrated competitive volumetric-bar-chart chip (CV-Chip) to assay multiple drug targets at the point-of-care (POC). To the best of our knowledge, it is the first time that a POC platform has been demonstrated to fully address the above-mentioned limitations. We applied this integrated CV-chip platform to assay multiple drugs in 38 patient urine and serum samples and validated the on-chip results with an LC-MS/MS method, indicating a clinical sensitivity and specificity of 0.94 and 1.00, respectively. We further demonstrated that the combination of an on-chip blood separator with the CV-Chip enabled the platform to directly assay finger-prick whole blood samples, which have always been recognized as an ideal biospecimen for POC detections. In summary, this integrated CV-Chip is able to serve as a sensitive, accurate, fast, portable, readout visible, and minimally invasive platform for drug abuse assessment.

Imaging of Cell-Cell Communication in a Vertical Orientation Reveals High-Resolution Structure of Immunological Synapse and Novel PD-1 Dynamics.

The immunological synapse (IS) is one of the most pivotal communication strategies in immune cells. Understanding the molecular basis of the IS provides critical information regarding how immune cells mount an effective immune response. Fluorescence microscopy provides a fundamental tool to study the IS. However, current imaging techniques for studying the IS cannot sufficiently achieve high resolution in real cell-cell conjugates. In this study, we present a new device that allows for high-resolution imaging of the IS with conventional confocal microscopy in a high-throughput manner. Combining micropits and single-cell trap arrays, we have developed a new microfluidic platform that allows visualization of the IS in vertically "stacked" cells. Using this vertical cell pairing (VCP) system, we investigated the dynamics of the inhibitory synapse mediated by an inhibitory receptor, programed death protein-1, and the cytotoxic synapse at the single-cell level. In addition to the technique innovation, we have demonstrated novel biological findings by this VCP device, including novel distribution of F-actin and cytolytic granules at the IS, programed death protein-1 microclusters at the NK IS, and kinetics of cytotoxicity. We propose that this high-throughput, cost-effective, easy-to-use VCP system, along with conventional imaging techniques, can be used to address a number of significant biological questions in a variety of disciplines.

Block-Cell-Printing for live single-cell printing.

A unique live-cell printing technique, termed "Block-Cell-Printing" (BloC-Printing), allows for convenient, precise, multiplexed, and high-throughput printing of functional single-cell arrays. Adapted from woodblock printing techniques, the approach employs microfluidic arrays of hook-shaped traps to hold cells at designated positions and directly transfer the anchored cells onto various substrates. BloC-Printing has a minimum turnaround time of 0.5 h, a maximum resolution of 5 µm, close to 100% cell viability, the ability to handle multiple cell types, and efficiently construct protrusion-connected single-cell arrays. The approach enables the large-scale formation of heterotypic cell pairs with controlled morphology and allows for material transport through gap junction intercellular communication. When six types of breast cancer cells are allowed to extend membrane protrusions in the BloC-Printing device for 3 h, multiple biophysical characteristics of cells--including the protrusion percentage, extension rate, and cell length--are easily quantified and found to correlate well with their migration levels. In light of this discovery, BloC-Printing may serve as a rapid and high-throughput cell protrusion characterization tool to measure the invasion and migration capability of cancer cells. Furthermore, primary neurons are also compatible with BloC-Printing.

Microfluidic Platforms for Yeast-Based Aging Studies.

The budding yeast Saccharomyces cerevisiae has been a powerful model for the study of aging and has enabled significant contributions to our understanding of basic mechanisms of aging in eukaryotic cells. However, the laborious low-throughput nature of conventional methods of performing aging assays limits the pace of discoveries in this field. Some of the technical challenges of conventional aging assay methods can be overcome by use of microfluidic systems coupled to time-lapse microscopy. One of the major advantages is the ability of a microfluidic system to perform long-term cell culture under well-defined environmental conditions while tracking individual yeast. Here, recent advancements in microfluidic platforms for various yeast-based studies including replicative lifespan assay, long-term culture and imaging, gene expression, and cell signaling are discussed. In addition, emerging problems and limitations of current microfluidic approaches are examined and perspectives on the future development of this dynamic field are presented.

Hypoxia induces a phase transition within a kinase signaling network in cancer cells.

Hypoxia is a near-universal feature of cancer, promoting glycolysis, cellular proliferation, and angiogenesis. The molecular mechanisms of hypoxic signaling have been intensively studied, but the impact of changes in oxygen partial pressure (pO2) on the state of signaling networks is less clear. In a glioblastoma multiforme (GBM) cancer cell model, we examined the response of signaling networks to targeted pathway inhibition between 21% and 1% pO2. We used a microchip technology that facilitates quantification of a panel of functional proteins from statistical numbers of single cells. We find that near 1.5% pO2, the signaling network associated with mammalian target of rapamycin (mTOR) complex 1 (mTORC1)--a critical component of hypoxic signaling and a compelling cancer drug target--is deregulated in a manner such that it will be unresponsive to mTOR kinase inhibitors near 1.5% pO2, but will respond at higher or lower pO2 values. These predictions were validated through experiments on bulk GBM cell line cultures and on neurosphere cultures of a human-origin GBM xenograft tumor. We attempt to understand this behavior through the use of a quantitative version of Le Chatelier's principle, as well as through a steady-state kinetic model of protein interactions, both of which indicate that hypoxia can influence mTORC1 signaling as a switch. The Le Chatelier approach also indicates that this switch may be thought of as a type of phase transition. Our analysis indicates that certain biologically complex cell behaviors may be understood using fundamental, thermodynamics-motivated principles.

Microfluidic Cell Deformability Assay for Rapid and Efficient Kinase Screening with the CRISPR-Cas9 System.

Herein we report a CRISPR-Cas9-mediated loss-of-function kinase screen for cancer cell deformability and invasive potential in a high-throughput microfluidic chip. In this microfluidic cell separation platform, flexible cells with high deformability and metastatic propensity flowed out, while stiff cells remained trapped. Through deep sequencing, we found that loss of certain kinases resulted in cells becoming more deformable and invasive. High-ranking candidates identified included well-reported tumor suppressor kinases, such as chk2, IKK-α, p38 MAPKs, and DAPK2. A high-ranking candidate STK4 was chosen for functional validation and identified to play an important role in the regulation of cell deformability and tumor suppression. Collectively, we have demonstrated that CRISPR-based on-chip mechanical screening is a potentially powerful strategy to facilitate systematic genetic analyses.

Competitive volumetric bar-chart chip with real-time internal control for point-of-care diagnostics.

Point-of-care (POC) testing has become widely used in clinical analysis because of its speed and portability; however, POC tools, such as lateral flow assays, suffer from low specificity, unclear readouts, and susceptibility to environmental and user errors. Herein, we report an ELISA-based competitive volumetric bar-chart chip (CV-chip) that eliminates these limitations. The CV-chip displays the readout in the form of ink bar charts based on direct competition between gases generated by the sample and the internal control. By employing a "competition mode", this platform decreases the potential influence of background resulting from environmental factors and provides visually clear positive or negative results without the requirement of calibration. In addition, the on-chip comparison enables the device to distinguish imperceptible differences (less than 1.3-fold) in human chorionic gonadotropin (hCG) concentrations that are near the cutoff value for pregnancy (∼1.4 ng/mL). We also utilized the ELISA-based CV-chip to successfully detect biomarkers from cancer cells. As a proof-of-concept application in a clinical setting, the CV-chip was employed to evaluate the status of drugs of abuse in 18 patients. For six different drugs, zero false-positive and very few false-negative (<2%) results were reported in more than 100 tests. This new ELISA platform offers a clinical diagnostics tool that is portable and easy to use, and provides improved clarity and sensitivity due to the inclusion of a real-time internal control.

Single-cell proteomic chip for profiling intracellular signaling pathways in single tumor cells.

We describe a microchip designed to quantify the levels of a dozen cytoplasmic and membrane proteins from single cells. We use the platform to assess protein-protein interactions associated with the EGF-receptor-mediated PI3K signaling pathway. Single-cell sensitivity is achieved by isolating a defined number of cells (n = 0-5) in 2 nL volume chambers, each of which is patterned with two copies of a miniature antibody array. The cells are lysed on-chip, and the levels of released proteins are assayed using the antibody arrays. We investigate three isogenic cell lines representing the cancer glioblastoma multiforme, at the basal level, under EGF stimulation, and under erlotinib inhibition plus EGF stimulation. The measured protein abundances are consistent with previous work, and single-cell analysis uniquely reveals single-cell heterogeneity, and different types and strengths of protein-protein interactions. This platform helps provide a comprehensive picture of altered signal transduction networks in tumor cells and provides insight into the effect of targeted therapies on protein signaling networks.