Immune-mediated inflammatory diseases and periodontal disease: a bidirectional two-sample mendelian randomization study.

BACKGROUND: Previous observational studies have shown a bidirectional association between immune-mediated inflammatory disorders (IMID) and periodontal disease. However, evidence regarding the causal role of IMID and periodontal disease is still lacking. Therefore, we conducted a bidirectional two-sample Mendelian randomization (MR) study to uncover the potential genetic causal effects between IMID and periodontal disease. METHODS: Bidirectional two-sample MR analysis was employed. Data for ten IMIDs were sourced from genome-wide association studies (GWAS) conducted by the FinnGen Consortium (range from 1023 to 36321 cases) and UK Biobank (UKB) (range from 150 to 17574 cases). Furthermore, GWAS data for periodontal disease were obtained from the FinnGen Consortium (87497 cases), UKB (458 cases), and Gene Lifestyle Interactions in Dental Endpoints (GLIDE) consortium (17,353 periodontitis cases). Subsequently, the causal relationships were analyzed by random effects inverse variance weighting, weighted median, and MR-Egger. Sensitivity analyses were performed using the Cochrane Q test, funnel plot, and Mr-Egger intercept test to ensure robustness. Eventually, replication analysis and meta-analysis across different databases were carried out. RESULTS: Systemic lupus erythematosus (SLE) [IVW: OR = 1.079 (95% CI: 1.032-1.128) and P < 0.001], Sjogren syndrome [IVW: OR = 1.082 (95% CI: 1.012-1.157) and P = 0.022] and hypothyroidism [IVW: OR = 1.52 (95% CI: 1.13-2.04) and P = 0.005] may increase the risk of periodontal disease. In addition, periodontal disease may reduce the risk of SLE [IVW: OR = 0.8079 (95% CI: 0.6764-0.9650) and P = 0.019] and hyperthyroidism [IVW: OR = 5.59*10-9 (95% CI: 1.43*10-15-2.18*10-2) and P = 0.014]. Meta-analysis indicated a causal correlation between SLE and an increased risk of periodontal disease: [OR = 1.08 (95% CI: 1.03-1.13), P = 0.0009]. No significant evidence suggests bilateral causal relationships between other IMIDs and periodontal disease. No significant estimation of heterogeneity or pleiotropy is detected. CONCLUSIONS: Our study has confirmed a genetic causal relationship between IMIDs and periodontal disease, thereby unveiling novel potential mechanisms underlying IMIDs and periodontal disease. This discovery is promising in fostering interdisciplinary collaboration between clinicians and stomatologists to facilitate appropriate and precise screening, prevention, and early treatment of IMIDs and periodontal disease.

Mechanisms underlying the therapeutic effects of isoflavones isolated from chickpea sprouts in treating osteoporosis based on network pharmacology.

Osteoporosis is a systemic bone disease that is caused by multiple factors that lead to an imbalance in bone metabolism. Isoflavones can prevent and treat osteoporosis by regulating bone metabolism through a variety of pathways. The germination of chickpeas can significantly increase their isoflavone contents. However, the use of isoflavones isolated from chickpea sprouts (ICS) to prevent and treat osteoporosis by regulating bone metabolism has not been widely studied. In vivo experimental studies in ovariectomized rats showed that ICS significantly improved femoral bone mineral density (BMD) and trabecular structure, with effects similar to raloxifene. Furthermore, the chemical composition of ICS as well as the targets and signalling pathways its regulates in the prevention and treatment of osteoporosis were predicted by network pharmacological studies. ICS with drug-like properties were identified by Lipinski's 5 principles, and intersecting targets of isoflavones with osteoporosis were identified. The overlapping targets were analysed by PPI, GO and KEGG analyses, and the possible key targets, signalling pathways and biological processes by which ICS treats osteoporosis were predicted; the prediction results were verified by molecular docking technology. The results showed that ICS could play an important role in the treatment of osteoporosis through "multicomponent, multitarget and multipathway" mechanisms, and the MAKP, NF-kB and ER-related signalling pathways may be important pathways by which ICS regulates osteoporosis; these findings provide a new theoretical basis for further experimental studies.

SMALPs Are Not Simply Nanodiscs: The Polymer-to-Lipid Ratios of Fractionated SMALPs Underline Their Heterogeneous Nature.

Amphipathic styrene-maleic acid (SMA) copolymers directly solubilize biomembranes into SMA-lipid particles, or SMALPs, that are often regarded as nanodiscs and hailed as a native membrane platform. The promising outlook of SMALPs inspires the discovery of many SMA-like copolymers that also solubilize biomembranes into putative nanodiscs, but a fundamental question remains on how much the SMALPs or SMALP analogues truly resemble the bilayer structure of nanodiscs. This unfortunate ambiguity undermines the utility of SMA or SMA-like copolymers in membrane biology because the structure and function of many membrane proteins depend critically on their surrounding matrices. Here, we report the structural heterogeneity of SMALPs revealed through fractionating SMALPs comprised of lipids and well-defined SMAs via size-exclusion chromatography followed by quantitative determination of the polymer-to-lipid (P/L) stoichiometric ratios in individual fractions. Through the lens of P/L stoichiometric ratios, different self-assembled polymer-lipid nanostructures are inferred, such as polymer-remodeled liposomes, polymer-encased nanodiscs, polymer-lipid mixed micelles, and lipid-doped polymer micellar aggregates. We attribute the structural heterogeneity of SMALPs to the microstructure variations amongst individual polymer chains that give rise to their polydisperse detergency. As an example, we demonstrate that SMAs with a similar S/MA ratio but different chain sizes participate preferentially in different polymer-lipid nanostructures. We further demonstrate that proteorhodopsin, a light-driven proton pump solubilized within the same SMALPs is distributed amongst different self-assembled nanostructures to display different photocycle kinetics. Our discovery challenges the native nanodisc notion of SMALPs or SMALP analogues and highlights the necessity to separate and identify the structurally dissimilar polymer-lipid particles in membrane biology studies.

Nanodiscs: a versatile nanocarrier platform for cancer diagnosis and treatment.

Cancer therapy is a significant challenge due to insufficient drug delivery to the cancer cells and non-selective killing of healthy cells by most chemotherapy agents. Nano-formulations have shown great promise for targeted drug delivery with improved efficiency. The shape and size of nanocarriers significantly affect their transport inside the body and internalization into the cancer cells. Non-spherical nanoparticles have shown prolonged blood circulation half-lives and higher cellular internalization frequency than spherical ones. Nanodiscs are desirable nano-formulations that demonstrate enhanced anisotropic character and versatile functionalization potential. Here, we review the recent development of theranostic nanodiscs for cancer mitigation ranging from traditional lipid nanodiscs encased by membrane scaffold proteins to newer nanodiscs where either the membrane scaffold proteins or the lipid bilayers themselves are replaced with their synthetic analogues. We first discuss early cancer detection enabled by nanodiscs. We then explain different strategies that have been explored to carry a wide range of payloads for chemotherapy, cancer gene therapy, and cancer vaccines. Finally, we discuss recent progress on organic-inorganic hybrid nanodiscs and polymer nanodiscs that have the potential to overcome the inherent instability problem of lipid nanodiscs.

Serum D-lactate, a novel serological biomarker, is promising for the diagnosis of periprosthetic joint infection.

BACKGROUND: Although many markers are used for diagnosis of periprosthetic joint infection (PJI), serological screening and diagnosis for PJI are still challenging. We evaluated the performance of serum D-lactate and compared it with ESR, coagulation-related biomarkers and synovial D-lactate for the diagnosis of PJI. METHODS: Consecutive patients with preoperative blood and intraoperative joint aspiration of a prosthetic hip or knee joint before revision arthroplasty were prospectively included. The diagnosis of PJI was based on the criteria of the Musculoskeletal Infection Society, and the diagnostic values of markers were estimated based on receiver operating characteristic (ROC) curves by maximizing sensitivity and specificity using optimal cutoff values. RESULTS: Of 52 patients, 26 (50%) were diagnosed with PJI, and 26 (50%) were diagnosed with aseptic failure. ROC curves showed that serum D-lactate, fibrinogen (FIB) and ESR had equal areas under the curve (AUCs) of 0.80, followed by D-dimer and fibrin degradation product, which had AUCs of 0.67 and 0.69, respectively. Serum D-lactate had the highest sensitivity of 88.46% at the optimal threshold of 1.14 mmol/L, followed by FIB and ESR, with sensitivities of 80.77% and 73.08%, respectively, while there were no significant differences in specificity (73.08%, 73.08% and 76.92%, respectively). CONCLUSION: Serum D-lactate showed similar performance to FIB and ESR for diagnosis of PJI. The advantages of serum D-lactate are pathogen-specific, highly sensitive, minimally invasive and rapidly available making serum D-lactate useful as a point-of-care screening test for PJI.

Grey matter alterations in restless legs syndrome: A coordinate-based meta-analysis.

Brain structural abnormalities in idiopathic restless legs syndrome have long been debated. Voxel-based morphometry is an objective structural magnetic resonance imaging technique to investigate regional grey matter volume or density differences between groups. In the last decade, voxel-based morphometry studies have exhibited inconsistent and conflicting findings regarding the presence and localization of brain grey matter alterations in restless legs syndrome. We therefore conducted a coordinate-based meta-analysis to quantitatively examine whether there were consistent grey matter findings in restless legs syndrome using the latest algorithms, seed-based d mapping with permutation of subject images. We included 12 voxel-based morphometry studies (13 datasets, 375 patients and 385 healthy controls). Our coordinate-based meta-analysis did not identify evidence of consistent grey matter alterations in restless legs syndrome. Grey matter alterations via voxel-based morphometry analysis are not therefore recommended to be used as a reliable surrogate neuroimaging marker for restless legs syndrome. This lack of consistency may be attributed to differences in sample size, genetics, gender distribution and age at onset, clinical heterogeneity (clinical course, anatomical distribution of symptoms, disease severity, disease duration, abnormal sensory profiles and comorbidity), and variations in imaging acquisition, data processing and statistical strategies. Longitudinal studies with multimodal neuroimaging techniques are needed to determine whether structural changes are dynamic and secondary to functional abnormalities.

Environmentally Benign Nanoantibiotics with a Built-in Deactivation Switch Responsive to Natural Habitats.

The massive use of antibiotics in healthcare and agriculture has led to their artificial accumulation in natural habitats, which risks the structure and function of the microbial communities in ecosystems, threatens food and water security, and accelerates the development of resistome. Ideally, antibiotics should remain fully active in clinical services while becoming deactivated rapidly once released into the environment, but none of the current antibiotics meet this criterion. Here, we show a nanoantibiotic design that epitomizes the concept of carrying a built-in "OFF" switch responsive to natural stimuli. The environmentally benign nanoantibiotics consist of cellulose backbones covalently grafted with hydrophilic polymer brushes that by themselves are antimicrobially inactive. In their nanostructured forms in services, these cellulose-based polymer molecular brushes are potent killers for both Gram-positive and Gram-negative bacteria, including clinical multidrug-resistant strains; after services and being discharged into the environment, they are shredded into antimicrobially inactive pieces by cellulases that do not exist in the human body but are abundant in natural habitats. This study illuminates a new concept of mitigating the environmental footprints of antibiotics with rationally designed nanoantibiotics that can be dismantled and disabled by bioorthogonal chemistry occurring exclusively in natural habitats.

Aberrant default mode network in amnestic mild cognitive impairment: a meta-analysis of independent component analysis studies.

Independent component analysis (ICA) is one of the most popular and valid methods to investigate the default mode network (DMN), an intrinsic network which attracts particular attention in amnestic mild cognitive impairment (aMCI). However, previous studies present inconsistent results regarding the topographical organization of the DMN in aMCI. Therefore, we conducted a quantitative, voxel-wise meta-analysis of resting-state ICA studies using Seed-based d Mapping to establish the most consistent pattern of DMN functional connectivity alterations in aMCI. Twenty studies, comprising 23 independent datasets involving 535 patients and 586 healthy controls, met the inclusion criteria. Patients with aMCI exhibited reliably lower DMN functional connectivity than the healthy controls in the bilateral precuneus/posterior cingulate cortices and medial temporal lobes, which are implicated in episodic memory deficits. Moreover, an exploratory meta-regression analysis revealed that greater severity of global cognitive impairment in the patient groups was associated with stronger functional connectivity in the bilateral medial frontal cortices (including the anterior cingulate cortices), left angular gyrus, and right temporal pole extending to the middle temporal gyrus, likely reflecting a compensatory mechanism for maintaining cognitive efficiency. This meta-analysis identifies a consistent pattern of aberrant DMN functional connectivity in aMCI, which facilitates understanding of the neurobiological substrates of this disease.

Two new triterpenes from Euphorbia alatavica.

A phytochemical investigation on Euphorbia alatavica Boiss resulted in the isolation of nine compounds, including two new ones, alatavolide and alatavoic acid (1-2). Chemical structures of these compounds were established on the basis of 1D, 2D NMR, and HR-MS techniques, and by comparison with data reported in the literature. Compounds 1, 2, 4, 6, 8, and 9 were screened for cytotoxicity using the MTT assay. Among these compounds, the new compound 2 showed moderate cytotoxicity against Hela, MCF-7 and A549 cell lines (IC50 values of 16.4 ± 3.2, 14.5 ± 2.8, 22.3 ± 3.1 µM, respectively), while the known compound 8 exhibited the most potent cytotoxicity with the IC50 values of 6.5 ± 3.1, 1.9 ± 0.9, 8.6 ± 3.5 µM, respectively.

Two new sesquiterpenoid glycosides from the leaves of Lycium barbarum.

Two new sesquiterpenoid glycosides, lyciumionosides A-B (1-2), together with four known compounds (3-6), were isolated from the leaves of Lycium barbarum. Their structures were mainly established on the basis of MS, 1D and 2D NMR spectroscopic techniques. The antiproliferative activities of compounds 1-5 were evaluated. Compound 1 showed highest inhibitory activity against A549 cells with IC50 value of 32.6 ± 2.6 µM, compound 3 showed highest inhibitory activity against PC-3 cells with IC50 value of 36.0 ± 2.9 µM, and compound 5 exhibited highest inhibitory activity against HeLa cells with IC50 value of 32.3 ± 4.2 µM.

Isoflavones extracted from chickpea Cicer arietinum L. sprouts induce mitochondria-dependent apoptosis in human breast cancer cells.

Isoflavones are important chemical components of the seeds and sprouts of chickpeas. We systematically investigated the effects of isoflavones extracted from chickpea sprouts (ICS) on the human breast cancer cell lines SKBr3 and Michigan Cancer Foundation-7 (MCF-7). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays showed that ICS (10-60 µg/mL) significantly inhibited the proliferation of both cell lines in a time-dependent and dose-dependent fashion. Wright-Giemsa staining as well as annexin V-fluorescein isothiocyanate and propidium iodide (Annexin V/PI) staining showed that ICS significantly increased cytoclasis and apoptotic body formation. Quantitative Annexin V/PI assays further showed that the number of apoptotic cells increased in a dose-dependent manner following ICS treatment. Semiquantitative reverse transcription PCR showed that ICS increased the expression of the apoptosis-promoting gene Bcl-2-associated X protein and decreased the expression of the antiapoptotic gene Bcl-2. Western blot analysis showed that treatment of SKBr3 and MCF-7 cells with ICS increased the expression of caspase 7, caspase 9, P53, and P21 in a dose-dependent manner. Flow cytometry assays using the fluorescent probe 3,3'-dihexyloxacarbocyanine iodide showed a dose-dependent decrease in mitochondrial membrane potential following ICS treatment. Treatment using ICS also induced a dose-dependent increase in reactive oxygen species production. This is the first study to demonstrate that ICS may be a chemopreventive or therapeutic agent against breast cancer.

Mn-ZIF nanozymes kill tumors by generating hydroxyl radical as well as reversing the tumor microenvironment.

Tumor tissues are well known for their unique high hydrogen peroxide (H2O2) microenvironment. How to exploit this tumor microenvironment for tumor cell killing is a question. In this study, a Mn-doped metal-organic framework (Mn-ZIF) was constructed. It possesses good peroxidase (POD) activity, which can oxidize tumor-localized H2O2 into hydroxyl radicals (·OH), that possesses the ability to directly kill tumor cells. More surprisingly, in vivo experiments the researchers not only observed the tumor-killing effect of Mn-ZIF, but also found it changes in macrophage phenotype in the tumor region. There was an increase in macrophage polarization towards the M1 subtype. This suggests that the tumor-killing effect of Mn-ZIF not only comes from its POD activity, but also regulates the immune microenvironment in the tumor region. In conclusion, the preparation of Mn-ZIF provides a new way for comprehensive tumor therapy.

CircRNA SEC24A promotes osteoarthritis through miR-107-5p/CASP3 axis.

Background: Osteoarthritis (OA) is the most frequently diagnosed chronic joint disease. CircSEC24A is significantly elevated in OA chondrocytes upon IL-1ß stimulation. However, its biological function in OA is still not fully understood. Methods: The circRNAs-miRNA-mRNA network was predicted by bioinformatics analysis. An in vitro OA chondrocytes model was established by IL-1ß stimulation. The expression of circSEC24A, miR-107-5p, CASP3, apoptosis-related molecules and extracellular matrix (ECM) components were detected by Western blot and qRT-PCR. MTT assay and Annexin V/PI staining were employed to monitor cell viability and apoptosis, respectively. The interaction between circSEC24A and miR-107-5p, as well as the binding between miR-107-5p and CASP3 3' UTR were detected by luciferase reporter and RIP assays. Cytokine secretion was monitored by ELISA assay. The role of circSEC24A was also explored in anterior cruciate ligament transection (ACLT) rat models. Results: CircSEC24A and CASP3 were increased, but miR-107-5p was decreased in rat OA cartilage tissues and OA chondrocytes. CircSEC24A acted as a sponge of miR-107-5p. Knockdown of circSEC24A promoted chondrocyte proliferation, but suppressed chondrocyte apoptosis, ECM degradation and inflammation via sponging miR-107-5p. CASP3 was identified as a miR-107-5p target gene. MiR-107-5p mimics protected against OA progression via targeting CASP3. Silencing of circSEC24A alleviated OA progression in ACLT model. Conclusion: CircSEC24A promotes OA progression through miR-107-5p/CASP3 axis.

Abnormal brain spontaneous neural activity in neuromyelitis optica spectrum disorder with neuropathic pain.

Background: Neuropathic pain is one of the most common symptoms in neuromyelitis optica spectrum disorder (NMOSD). Notwithstanding, its underlying mechanism remains obscure. Methods: The amplitude of low-frequency fluctuations (ALFF) metric was employed to investigate spontaneous neural activity alterations via resting-state functional magnetic resonance imaging (rs-MRI) data from a 3.0 T MRI scanner, in a sample of 26 patients diagnosed with NMOSD with neuropathic pain (NMOSD-WNP), 20 patients with NMOSD but without neuropathic pain (NMOSD-WoNP), and 38 healthy control (HC) subjects matched for age and sex without the comorbidity of depressive or anxious symptoms. Results: It was observed that patients with NMOSD-WNP displayed a significant ALFF decrease in the left amygdala and right anterior insula, relative to both patients with NMOSD-WoNP and HC subjects. Furthermore, ALFF values in the left amygdala were negatively correlated with the scores of the Douleur Neuropathique en 4 Questions and McGill Pain Questionnaire (both sensory and affective descriptors) in patients with NMOSD-WNP. Additionally, there were negative correlations between the ALFF values in the right anterior insula and the duration of pain and the number of relapses in patients with NMOSD-WNP. Conclusion: The present study characterizes spontaneous neural activity changes in brain regions associated with sensory and affective processing of pain and its modulation, which underscore the central aspects in patients with NMOSD-WNP. These findings might contribute to a better understanding of the pathophysiologic basis of neuropathic pain in NMOSD.

Isoflavones isolated from chickpea sprouts alleviate ovariectomy-induced osteoporosis in rats by dual regulation of bone remodeling.

Osteoporosis is a common systemic skeletal disease and a predominant underlying factor in the increased occurrence of fractures. The structure of isoflavones resembles that of estrogen and can confer similar but weaker effects. This study investigated the potential inhibitory effects of isoflavones from chickpea sprouts (ICS) on ovariectomy (OVX)-induced osteoporosis in vitro and in vivo. Notably, we found that ICS treatment could attenuate bone loss and improve trabecular microarchitecture and biomechanical properties of the fourth lumbar vertebra in OVX-induced osteoporotic rats and could also inhibit the development of a hyperosteometabolic state in this model. The osteogenic differentiation of bone marrow stem cells (BMSCs) was significantly enhanced by ICS intervention in vitro, and we confirmed that estrogen receptor α signaling was required for this increased osteogenic differentiation. Additionally, ICS has been shown to inhibit bone resorption via ERa modulation of the OPG/RANKL pathway. RANKL-induced osteoclastogenesis was reduced under ICS treatment, supporting that NF-κB signaling was inhibited by ICS. Thus, ICS attenuates osteoporosis progression by promoting osteogenic differentiation and inhibiting osteoclastic resorption. These results support the further exploration and development of ICS as a pharmacological agent for the treatment and prevention of osteoporosis.

Assessment of the estrogenic activities of chickpea (Cicer arietinum L) sprout isoflavone extract in ovariectomized rats.

AIM: Chickpea (Cicer arietinum L) is a traditional Uighur herb. In this study we investigated the estrogenic activities of the isoflavones extracted from chickpea sprouts (ICS) in ovariectomized rats. METHODS: Ten-week-old virgin Sprague-Dawley female rats were ovariectomized (OVX). The rats were administered via intragastric gavage 3 different doses of ICS (20, 50, or 100 mg·kg(-1)·d(-1)) for 5 weeks. Their uterine weight and serum levels of 17ß-estradiol (E2), follicle stimulating hormone (FSH) and luteinizing hormone (LH) were measured. The epithelial height, number of glands in the uterus, and number of osteoclasts in the femur were histologically quantified, and the expression of proliferating cell nuclear antigen (PCNA) was assessed immunohistochemically. Bone structural parameters, including bone mineral density (BMD), bone volume/tissue volume (BV/TV), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp) were measured using Micro-CT scanning. RESULTS: Treatments of OVX rats with ICS (50 or 100 mg·kg(-1)·d(-1)) produced significant estrogenic effects on the uteruses, including the increases in uterine weight, epithelial height and gland number, as well as in the expression of the cell proliferation marker PCNA. The treatments changed the secretory profile of ovarian hormones and pituitary gonadotropins: serum E2 level was significantly increased, while serum LH and FSH levels were decreased compared with the vehicle-treated OVX rats. Furthermore, the treatments significantly attenuated the bone loss, increased BMD, BV/TV and Tb.Th and decreased Tb.Sp and the number of osteoclasts. Treatment of OVX rats with the positive control drug E2 (0.25 mg·kg(-1)·d(-1)) produced similar, but more prominent effects. CONCLUSION: ICS exhibits moderate estrogenic activities as compared to E2 in ovariectomized rats, suggesting the potential use of ICS for the treatment of menopausal symptoms and osteoporosis caused by estrogen deficiency.

Harnessing extremophilic carboxylesterases for applications in polyester depolymerisation and plastic waste recycling.

The steady growth in industrial production of synthetic plastics and their limited recycling have resulted in severe environmental pollution and contribute to global warming and oil depletion. Currently, there is an urgent need to develop efficient plastic recycling technologies to prevent further environmental pollution and recover chemical feedstocks for polymer re-synthesis and upcycling in a circular economy. Enzymatic depolymerization of synthetic polyesters by microbial carboxylesterases provides an attractive addition to existing mechanical and chemical recycling technologies due to enzyme specificity, low energy consumption, and mild reaction conditions. Carboxylesterases constitute a diverse group of serine-dependent hydrolases catalysing the cleavage and formation of ester bonds. However, the stability and hydrolytic activity of identified natural esterases towards synthetic polyesters are usually insufficient for applications in industrial polyester recycling. This necessitates further efforts on the discovery of robust enzymes, as well as protein engineering of natural enzymes for enhanced activity and stability. In this essay, we discuss the current knowledge of microbial carboxylesterases that degrade polyesters (polyesterases) with focus on polyethylene terephthalate (PET), which is one of the five major synthetic polymers. Then, we briefly review the recent progress in the discovery and protein engineering of microbial polyesterases, as well as developing enzyme cocktails and secreted protein expression for applications in the depolymerisation of polyester blends and mixed plastics. Future research aimed at the discovery of novel polyesterases from extreme environments and protein engineering for improved performance will aid developing efficient polyester recycling technologies for the circular plastics economy.

High-throughput examination of fluorescence resonance energy transfer-detected metal-ion response in mammalian cells.

Fluorescence resonance energy transfer (FRET)-based genetically encoded metal-ion sensors are important tools for studying metal-ion dynamics in live cells. We present a time-resolved microfluidic flow cytometer capable of characterizing the FRET-based dynamic response of metal-ion sensors in mammalian cells at a throughput of 15 cells/s with a time window encompassing a few milliseconds to a few seconds after mixing of cells with exogenous ligands. We have used the instrument to examine the cellular heterogeneity of Zn(2+) and Ca(2+) sensor FRET response amplitudes and demonstrated that the cluster maps of the Zn(2+) sensor FRET changes resolve multiple subpopulations. We have also measured the in vivo sensor response kinetics induced by changes in Zn(2+) and Ca(2+) concentrations. We observed an ∼30 fold difference between the extracellular and intracellular sensors.