Molecular basis of differential receptor usage for naturally occurring CD55-binding and -nonbinding coxsackievirus B3 strains.

Receptor usage defines cell tropism and contributes to cell entry and infection. Coxsackievirus B (CVB) engages coxsackievirus and adenovirus receptor (CAR), and selectively utilizes the decay-accelerating factor (DAF; CD55) to infect cells. However, the differential receptor usage mechanism for CVB remains elusive. This study identified VP3-234 residues (234Q/N/V/D/E) as critical population selection determinants during CVB3 virus evolution, contributing to diverse binding affinities to CD55. Cryoelectron microscopy (cryo-EM) structures of CD55-binding/nonbinding isolates and their complexes with CD55 or CAR were obtained under both neutral and acidic conditions, and the molecular mechanism of VP3-234 residues determining CD55 affinity/specificity for naturally occurring CVB3 strains was elucidated. Structural and biochemical studies in vitro revealed the dynamic entry process of CVB3 and the function of the uncoating receptor CAR with different pH preferences. This work provides detailed insight into the molecular mechanism of CVB infection and contributes to an in-depth understanding of enterovirus attachment receptor usage.

Lysyl hydroxylase LH1 promotes confined migration and metastasis of cancer cells by stabilizing Septin2 to enhance actin network.

BACKGROUND: Excessive extracellular matrix deposition and increased stiffness are typical features of solid tumors such as hepatocellular carcinoma (HCC) and pancreatic ductal adenocarcinoma (PDAC). These conditions create confined spaces for tumor cell migration and metastasis. The regulatory mechanism of confined migration remains unclear. METHODS: LC-MS was applied to determine the differentially expressed proteins between HCC tissues and corresponding adjacent tissue. Collective migration and single cell migration microfluidic devices with 6 µm-high confined channels were designed and fabricated to mimic the in vivo confined space. 3D invasion assay was created by Matrigel and Collagen I mixture treat to adherent cells. 3D spheroid formation under various stiffness environment was developed by different substitution percentage GelMA. Immunoprecipitation was performed to pull down the LH1-binding proteins, which were identified by LC-MS. Immunofluorescent staining, FRET, RT-PCR, Western blotting, FRAP, CCK-8, transwell cell migration, wound healing, orthotopic liver injection mouse model and in vivo imaging were used to evaluate the target expression and cellular phenotype. RESULTS: Lysyl hydroxylase 1 (LH1) promoted the confined migration of cancer cells at both collective and single cell levels. In addition, LH1 enhanced cell invasion in a 3D biomimetic model and spheroid formation in stiffer environments. High LH1 expression correlated with poor prognosis of both HCC and PDAC patients, while it also promoted in vivo metastasis. Mechanistically, LH1 bound and stabilized Septin2 (SEPT2) to enhance actin polymerization, depending on the hydroxylase domain. Finally, the subpopulation with high expression of both LH1 and SEPT2 had the poorest prognosis. CONCLUSIONS: LH1 promotes the confined migration and metastasis of cancer cells by stabilizing SEPT2 and thus facilitating actin polymerization.

High Throughput Confined Migration Microfluidic Device for Drug Screening.

Cancer metastasis is the major cause of cancer-related death. Excessive extracellular matrix deposition and increased stiffness are typical features of solid tumors, creating confined spaces for tumor cell migration and metastasis. Confined migration is involved in all metastasis steps. However, confined and unconfined migration inhibitors are different and drugs available to inhibit confined migration are rare. The main challenges are the modeling of confined migration, the suffering of low throughput, and others. Microfluidic device has the advantage to reduce reagent consumption and enhance throughput. Here, a microfluidic chip that can achieve multi-function drug screening against the collective migration of cancer cells under confined environment is designed. This device is applied to screen out effective drugs on confined migration among a novel mechanoreceptors compound library (166 compounds) in hepatocellular carcinoma, non-small lung cancer, breast cancer, and pancreatic ductal adenocarcinoma cells. Three compounds that can significantly inhibit confined migration in pan-cancer: mitochonic acid 5 (MA-5), SB-705498, and diphenyleneiodonium chloride are found. Finally, it is elucidated that these drugs targeted mitochondria, actin polymerization, and cell viability, respectively. In sum, a high-throughput microfluidic platform for screening drugs targeting confined migration is established and three novel inhibitors of confined migration in multiple cancer types are identified.

Molecular Basis of Mink ACE2 Binding to SARS-CoV-2 and Its Mink-Derived Variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is transmitted between humans and minks, and some mutations in the spike (S) protein, especially in the receptor-binding domain (RBD), have been identified in mink-derived viruses. Here, we examined binding of the mink angiotensin-converting enzyme 2 (ACE2) receptor to mink-derived and important human-originating variants, and we demonstrated that most of the RBD variants increased the binding affinities to mink ACE2 (mkACE2). Cryo-electron microscopy structures of the mkACE2-RBD Y453F (with a Y-to-F change at position 453) and mkACE2-RBD F486L complexes helped identify the key residues that facilitate changes in mkACE2 binding affinity. Additionally, the data indicated that the Y453F and F486L mutations reduced the binding affinities to some human monoclonal antibodies, and human vaccinated sera efficiently prevented infection of human cells by pseudoviruses expressing Y453F, F486L, or N501T RBD. Our findings provide an important molecular mechanism for the rapid adaptation of SARS-CoV-2 in minks and highlight the potential influence of the main mink-originating variants for humans. IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a broad range of hosts. Mink-derived SARS-CoV-2 can transmit back to humans. There is an urgent need to understand the binding mechanism of mink-derived SARS-CoV-2 variants to mink receptor. In this study, we identified all mutations in the receptor-binding domain (RBD) of spike (S) protein from mink-derived SARS-CoV-2, and we demonstrated the enhanced binding affinity of mink angiotensin-converting enzyme 2 (ACE2) to most of the mink-derived RBD variants as well as important human-originating RBD variants. Cryo-electron microscopy structures revealed that the Y453F and F486L mutations enhanced the binding forces in the interaction interface. In addition, Y453F and F486L mutations reduced the binding affinities to some human monoclonal antibodies, and the SARS-CoV-2 pseudoviruses with Y453F, F486L, or N501T mutations were neutralized by human vaccinated sera. Therefore, our results provide valuable information for understanding the cross-species transmission mechanism of SARS-CoV-2.

An integrated workflow for biomarker development using microRNAs in extracellular vesicles for cancer precision medicine.

EV-miRNAs are microRNA (miRNA) molecules encapsulated in extracellular vesicles (EVs), which play crucial roles in tumor pathogenesis, progression, and metastasis. Recent studies about EV-miRNAs have gained novel insights into cancer biology and have demonstrated a great potential to develop novel liquid biopsy assays for various applications. Notably, compared to conventional liquid biomarkers, EV-miRNAs are more advantageous in representing host-cell molecular architecture and exhibiting higher stability and specificity. Despite various available techniques for EV-miRNA separation, concentration, profiling, and data analysis, a standardized approach for EV-miRNA biomarker development is yet lacking. In this review, we performed a substantial literature review and distilled an integrated workflow encompassing important steps for EV-miRNA biomarker development, including sample collection and EV isolation, EV-miRNA extraction and quantification, high-throughput data preprocessing, biomarker prioritization and model construction, functional analysis, as well as validation. With the rapid growth of "big data", we highlight the importance of efficient mining of high-throughput data for the discovery of EV-miRNA biomarkers and integrating multiple independent datasets for in silico and experimental validations to increase the robustness and reproducibility. Furthermore, as an efficient strategy in systems biology, network inference provides insights into the regulatory mechanisms and can be used to select functionally important EV-miRNAs to refine the biomarker candidates. Despite the encouraging development in the field, a number of challenges still hinder the clinical translation. We finally summarize several common challenges in various biomarker studies and discuss potential opportunities emerging in the related fields.

Development of a carboxyl-terminated indium tin oxide electrode for improving cell adhesion and facilitating low noise, real-time impedance measurements.

The working electrode's surface property is crucial to cell adhesion and signal collection in electric cell-substrate impedance sensing (ECIS). To date, the indium tin oxide (ITO)-based working electrode is of interest in ECIS study due to its high transparency and biocompatibility. Of great concern is the impedance signal loss, distortion, and data interpretation conflict profoundly created by the movement of multiple cells during ECIS study. Here, a carboxyl-terminated ITO substrate was prepared by stepwise surface amino silanization, with N-hydroxy succinimide (NHS) and 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC) treatment, respectively. We investigated the stepwise changes in the property of the treated ITO, cell-substrate adhesion, collective cell mobility, and time course of change in absolute impedance from multiple Chinese hamster ovary (CHO) cells [(Δt-Δ|Z|)CELLS]. The carboxyl-terminated ITO substrate with a surface roughness of 6.37 nm shows enhanced conductivity, 75% visible light transparency, improved cell adherence, reduced collective cell migration speed by approximately twofold, and diminished signal distortion in the [(Δt-Δ|Z|)CELLS]. Thus, our study provides an ITO surface-treatment strategy to reduce multiple cell movement effects and to obtain essential cell information from the ECIS study of multiple cells through undistorted (Δt-Δ|Z|)CELLS.

FROZEN! Intracellular multi-electrolyte analysis measures millimolar lithium in mammalian cells.

Lithium salts are commonly used as medication for Bipolar Disorder (BD) and depression. However, there are limited methods to quantify intracellular lithium. Most methods to analyze intracellular electrolytes require tedious sample processing, specialized and often expensive machinery, sometimes involving harmful chemicals, and a bulk amount of the sample. In this work, we report a novel method (FROZEN!) based on cell isolation (from the surrounding medium) through rapid de-ionized water cleaning, followed by flash freezing for preservation. SKOV3 cells were cultured in normal medium and a medium containing 1.0 mM lithium. Lithium and other intracellular electrolytes in the isolated and preserved cells were simultaneously analyzed with laser-induced breakdown spectroscopy (LIBS) and X-ray fluorescence spectroscopy (XRF). Key electrolytes such as sodium, potassium, and magnesium, along with lithium, were detectable at the single-cell level. We found that cells cultured in the lithium medium have an intracellular lithium concentration of 0.5 mM. Concurrently, the intracellular concentrations of other positively charged electrolytes (sodium, potassium, and magnesium) were reduced by the presence of lithium. FROZEN! will greatly facilitate research in intracellular electrolyte balance during drug treatment, or other physiological stresses. In particular, the cell isolation and preservation steps can be easily performed by many laboratories worldwide, after which the samples are sent to an analytical laboratory for electrolyte analysis.

Dissecting the novel partners of nuclear c-Raf and its role in all-trans retinoic acid (ATRA)-induced myeloblastic leukemia cells differentiation.

All-trans retinoic acid (ATRA) is an anti-cancer differentiation therapy agent effective for acute promyelocytic leukemia (APL) but not acute myeloid leukemia (AML) in general. Using the HL-60 human non-APL AML model where ATRA causes nuclear enrichment of c-Raf that drives differentiation and G1/G0 cell cycle arrest, we now observe that c-Raf in the nucleus showed novel interactions with several prominent regulators of the cell cycle and cell differentiation. One is cyclin-dependent kinase 2 (Cdk2). ATRA treatment caused c-Raf to dissociate from Cdk2. This was associated with enhanced binding of Cdk2 with retinoic acid receptor α (RARα). Consistent with this novel Raf/CDK2/RARα axis contributing to differentiation, CD38 expression per cell, which is transcriptionally regulated by a retinoic acid response element (RARE), is enhanced. The RB tumor suppressor, a fundamental regulator of G1 cell cycle progression or arrest, was also targeted by c-Raf in the nucleus. RB and specifically the S608 phosphorylated form (pS608RB) complexed with c-Raf. ATRA treatment induced S608RB-hypophosphorylation associated with G1/G0 cell cycle arrest and release of c-Raf from RB. We also found that nuclear c-Raf interacted with SMARCD1, a pioneering component of the SWI/SNF chromatin remodeling complex. ATRA treatment diminished the amount of this protein bound to c-Raf. The data suggest that ATRA treatment to HL-60 human cells re-directed c-Raf from its historically pro-proliferation functions in the cytoplasm to pro-differentiation functions in the nucleus.

PEG-PtS2 nanosheet-based fluorescence biosensor for label-free human papillomavirus genotyping.

A simple and efficient ultrasonication-assisted liquid exfoliation method is proposed to produce PtS2 nanosheets on a large scale and improve their dispersion in aqueous solution by surface polyethylene glycol modification. The interaction of polyethylene glycol-modified PtS2 (PEG-PtS2) nanosheets with fluorescent labeled DNA and the fluorescence quenching mechanism using FAM-labeled hpv16e6 gene fragment as a probe was investigated. The excitation and emission wavelengths were 468 and 517 nm, respectively. The fluorescence quenching mechanism of PEG-PtS2 nanosheets for double-stranded DNA (dsDNA) might stem from the static quenching effect. Based on the difference in fluorescence quenching capability of PEG-PtS2 nanosheets in fluorescent probe tagged single-stranded DNA (ssDNA) and dsDNA, a mix-and-detect method was proposed for determination of DNA. Without the need for probe immobilization and tedious washing steps, the genotyping of human papillomavirus (HPV) was easily achieved. The limit of detection was calculated to 0.44 nM, showing a good linear range within 0.05-10 nM. We believe this biosensor provides opportunities to develop a simple and low-cost strategy for molecular diagnostics. Graphical abstract.

Autophagy inhibitor Vacuolin-1 interferes with lipid-based small interference RNA delivery.

Autophagy and endocytosis are important pathways regulating macromolecule recycling and regeneration. Small molecule inhibitors are utilized to modulate these pathways and to treat autophagy-related diseases. Vacuolin-1 is a small molecule that can potently and reversibly inhibit autophagy by activating Rab5. In addition, Vacuolin-1 can be applied to inhibit exocytosis in a variety of cell types. Here we report that Vacuolin-1 significantly reduces small interference RNA (siRNA)-mediated gene silencing delivered by liposome transfection reagent or lipid nanoparticles in Hela cells. Vacuolin-1 exhibits the strongest inhibition effect among a few autophagy inhibitors including Chloroquine, Wortmannin, and Bafilomycin A1. We found that siRNAs are over-accumulated intracellularly and colocalized with a late endosome marker Rab7 in Vacuolin-1 treated cells, suggesting Vacuolin-1 inhibits the cytoplasmic release of lipid siRNA complexes from late endosomes. We propose that Vacuolin-1 could potentially be used to control the effects of lipid nanoparticle-based RNAi and gene therapy drugs.

Facile Synthesis of Gadolinium Chelate-Conjugated Polymer Nanoparticles for Fluorescence/Magnetic Resonance Dual-Modal Imaging.

Fluorescence (FL)/magnetic resonance (MR) dual-modal imaging nanoprobes are significant not only for cutting edge research in molecular imaging, but also for clinical diagnosis with high precision and accuracy. However, synthesis of FL/MR dual-modal imaging nanoprobes that simultaneously exhibit strong fluorescent brightness and high MR response, long-term colloidal stability with uniform sizes, good biocompatibility and a versatile surface functionality has proven challenging. In this study, the well-defined core-shell structured Gd3+ chelate-conjugated fluorescent polymer nanoparticles (Gd-FPNPs) that consist of rhodamine B (RB)-encapsulated poly(methyl methacrylate) (PMMA) cores and Gd3+ chelate-conjugated branched polyethylenimine (PEI) shells, are facilely synthesized via a one-step graft copolymerization of RB-encapsulated MMA from PEI-DTPA-Gd induced by tert-butyl hydroperoxide (TBHP) at 80 °C for 2 h. The mild synthesis route not only preserves the chemical environment for Gd3+ coordination, but also improves optical properties and chemo-/photostability of RB. A high local concentration of outer surface-chelated Gd3+ and their direct interactions with hydrogen protons endow Gd-FPNPs high longitudinal relaxivity (26.86 mM-1 s-1). The uniform spherical structure of Gd-FPNPs facilitates their biotransfer, and their surface carboxyl and amine groups afford them both long-term colloidal stability and cell-membrane permeability. The excellent biocompatibility and FL/MR dual-modal imaging capability of Gd-FPNPs are demonstrated using HeLa cells and mice as models. All the results confirm that Gd-FPNPs fulfill the design criteria for a high-performance imaging nanoprobe. In addition, this study enables such probes to be prepared also by those not skilled in nanomaterial synthesis, and thus promoting the development of novel functional imaging nanoprobes.

Label-free detection of live cancer cells and DNA hybridization using 3D multilayered plasmonic biosensor.

Three-dimensional (3D) multilayered plasmonic nanostructures consisting of Au nanosquares on top of SU-8 nanopillars, Au asymmetrical nanostructures in the middle, and Au asymmetrical nanoholes at the bottom were fabricated through reversal nanoimprint technology. Compared with two-dimensional and quasi-3D plasmonic nanostructures, the 3D multilayered plasmonic nanostructures showed higher electromagnetic field intensity, longer plasmon decay length and larger plasmon sensing area, which are desirable for highly sensitive localized surface plasmonic resonance biosensors. The sensitivity and resonance peak wavelength of the 3D multilayered plasmonic nanostructures could be adjusted by varying the offset between the top and bottom SU-8 nanopillars from 31% to 56%, and the highest sensitivity of 382 and 442 nm/refractive index unit were observed for resonance peaks at 581 and 805 nm, respectively. Live lung cancer A549 cells with a low concentration of 5 × 103 cells ml-1 and a low sample volume of 2 µl could be detected by the 3D multilayered plasmonic nanostructures integrated in a microfluidic system. The 3D plasmonic biosensors also had the advantages of detecting DNA hybridization by capturing the complementary target DNA in the low concentration range of 10-14-10-7 M, and providing a large peak shift of 82 nm for capturing 10-7 M complementary target DNA without additional signal amplification.

Effects of direct current electric fields on lung cancer cell electrotaxis in a PMMA-based microfluidic device.

Tumor metastasis is the primary cause of cancer death. Numerous studies have demonstrated the electrotactic responses of various cancer cell types, and suggested its potential implications in metastasis. In this study, we used a microfluidic device to emulate endogenous direct current electric field (dcEF) environment, and studied the electrotactic migration of non-small cell lung cancer cell lines (H460, HCC827, H1299, and H1975) and the underlying mechanisms. These cell lines exhibited greatly different response in applied dcEFs (2-6 V/cm). While H460 cells (large cell carcinoma) showed slight migration toward cathode, H1299 cells (large cell carcinoma) showed increased motility and dcEF-dependent anodal migration with cell reorientation. H1975 cells (adenocarcinoma) showed dcEF-dependent cathodal migration with increased motility, and HCC827 cells (adenocarcinoma) responded positively in migration speed and reorientation but minimally in migrating directions to dcEF. Activation of MAPK and PI3K signaling pathways was found to be associated with the realignment and directed migration of lung cancer cells. In addition, both Ca2+ influx through activated stretch-activated calcium channels (SACCs) (but not voltage-gated calcium channels, VGCCs) and Ca2+ release from intracellular storage were involved in lung cancer cell electrotactic responses. The results demonstrated that the microfluidic device provided a stable and controllable microenvironment for cell electrotaxis study, and revealed that the electrotactic responses of lung cancer cells were heterogeneous and cell-type dependent, and multiple signals contributed to lung cancer cells electrotaxis.

Icariin Stimulates Differentiation and Suppresses Adipocytic Transdifferentiation of Primary Osteoblasts Through Estrogen Receptor-Mediated Pathway.

Icariin, the main constituent of Herba Epimedii, appears to be a promising alternative to classic drugs used to treat osteoporosis. However, the detailed molecular mechanisms of its action and the role of icariin in the cross-talk between osteoblasts and adipocytes remain unclear. The present study was designed to investigate the gene expression profile of primary osteoblasts in the presence of icariin, and the effects of icariin on the differentiation and adipogenic transdifferentiation of osteoblasts. Cellular and molecular markers expressed during osteoblastic differentiation were assessed by cytochemical analysis, real-time quantitative PCR, Western blotting, and cDNA microarray analysis. Results indicated that icariin up-regulated the expression of runt-related transcription factor 2 (Runx2), bone morphogenetic protein 2 (Bmp2), and collagen type 1 (Col1) genes, and down-regulated the expression of the peroxisome proliferator-activated receptor γ (Pparg) and CCAAT/enhancer-binding protein ß (Cebpb) genes. These effects were blocked by ICI 182,780, suggesting that icariin may be acting via the estrogen receptor (ER). Results also demonstrated that the ratio of osteoprotegerin (Opg)/receptor activator of nuclear factor kappa B ligand (Rankl) expression was up-regulated following treatment with icariin. In total, osteoblastic gene expression profile analysis suggested that 33 genes were affected by icariin; these could be sub-divided into nine functional categories. It appears that icariin could stimulate the differentiation and mineralization of osteoblasts, regulate the differentiation of osteoclasts, and inhibit the adipogenic transdifferentiation of osteoblasts, therefore increasing the number of osteoblasts undergoing differentiation to mature osteoblasts, via an ER-mediated pathway. In summary, icariin may exhibit beneficial effects on bone health, especially for patients with osteoporosis and obesity.

High sensitivity plasmonic biosensor based on nanoimprinted quasi 3D nanosquares for cell detection.

Quasi three-dimensional (3D) plasmonic nanostructures consisting of Au nanosquares on top of SU-8 nanopillars and Au nanoholes on the bottom were developed and fabricated using nanoimprint lithography with simultaneous thermal and UV exposure. These 3D plasmonic nanostructures were used to detect cell concentration of lung cancer A549 cells, retinal pigment epithelial (RPE) cells, and breast cancer MCF-7 cells. Nanoimprint technology has the advantage of producing high uniformity plasmonic nanostructures for such biosensors. Multiple resonance modes were observed in these quasi 3D plasmonic nanostructures. The hybrid coupling of localized surface plasmon resonances and Fabry-Perot cavity modes in the quasi 3D nanostructures resulted in high sensitivity of 496 nm/refractive index unit. The plasmonic resonance peak wavelength and sensitivity could be tuned by varying the Au thickness. Resonance peak shifts for different cells at the same concentration were distinct due to their different cell area and confluency. The cell concentration detection limit covered a large range of 5 × 10(2) to 1 × 10(7) cells ml(-1) with these new plasmonic nanostructures. They also provide a large resonance peak shift of 51 nm for as little as 0.08 cells mm(-2) of RPE cells for high sensitivity cell detection.

Two chiroptical modes of silver nanospirals.

As an emerging three-dimensional chiral metamaterial, plasmonic nanospirals (NSs) possess inherent chiroptical activity that has attracted increasing attention for developing potential photonic applications. However, the study of chiroptical activity of plasmonic NSs is still in its infancy, especially for NSs made of silver, which is a typical plasmonic material with high plasmonic quality. Herein, we present a systematic study of circular dichroism (CD) of silver NSs (AgNSs) that are fabricated and engineered in helical lengths by glancing-angle deposition (GLAD) and dispersed in ethanol. The CD spectrum is composed of a bisignated mode of two peaks, one in the UV regime and the other in the visible. The UV mode has a resonance wavelength saturating at ∼375 nm and a linewidth decoupled from the helical elongation, while the visible mode tends to have a redshift and its linewidth broadens linearly with the elongation of AgNS. Helical elongation generally amplifies the chiroptical activity of both modes. Finite-element simulation shows good agreement with the experimental CD results, and accounts for the wavelength-related chiroptical distinction in terms of the resonance wavelength. This work contributes to understanding the bisignated chiroptical responses of plasmonic nanospirals, and introduces a simple method to tailor the visible chiroptical activity that is strongly desired to explore a wide range of chirality-related bio-applications.

Au Nanoparticles Decorated TiO2 Nanotube Arrays as a Recyclable Sensor for Photoenhanced Electrochemical Detection of Bisphenol A.

A photorefreshable and photoenhanced electrochemical sensing platform for bisphenol A (BPA) detection based on Au nanoparticles (NPs) decorated carbon doped TiO2 nanotube arrays (TiO2/Au NTAs) is described. The TiO2/Au NTAs were prepared by quick annealing of anodized nanotubes in argon, followed by controllable electrodeposition of Au NPs. The decoration of Au NPs not only improved photoelectrochemical behavior but also enhanced electrocatalytic activities of the resulted hybrid NTAs. Meanwhile, the high photocatalytic activity of the NTAs allowed the electrode to be readily renewed without damaging the microstructures and surface states after a short UV treatment. The electrochemical detection of BPA on TiO2/Au NTAs electrode was significantly improved under UV irradiation as the electrode could provide fresh reaction surface continuously and the further increased photocurrent resulting from the improved separation efficiency of the photogenerated electron-hole pairs derived from the consumption of holes by BPA. The results showed that the refreshable TiO2/Au NTAs electrode is a promising sensor for long-term BPA monitoring with the detection limit (S/N = 3) of 6.2 nM and the sensitivity of 2.8 µA·µM(-1)·cm(-2).

Flavonoids of Herba Epimedii stimulate osteogenic differentiation and suppress adipogenic differentiation of primary mesenchymal stem cells via estrogen receptor pathway.

CONTEXT: Accumulating evidence indicates that Herba Epimedii [Epimedii folium (Berberidaceae)] has anti-osteoporotic effect by stimulating osteoblastic bone formation and reducing osteoclastic bone resorption. However, the effect of Herba Epimedii in regulating the cross-talk between osteogenic and adipogenic differentiation of mesenchymal stem cells (MSCs) remains unclear. OBJECTIVE: The present study investigates the effect of total flavonoids of Herba Epimedii (HETF) on the osteogenesis and adipogenesis of primary MSCs. MATERIALS AND METHODS: HETF were prepared and identified by HPLC-fingerprinting, primary mouse MSCs in the presence of 0.006-6 µg/mL HETF for 2-10 d were subject to morphological, biochemical, and quantitative real-time PCR analysis. RESULTS: Sixteen chemical components were identified in HETF by HPLC-fingerprinting and account for over 95% of the total area of HPLC peaks. During osteogenesis of MSCs, 0.006-6 µg/mL HETF promoted the proliferation of MSCs from 17% to 22%, increased alkaline phosphatase activity up to 3.7-fold (0.6 µg/mL), and extracellular calcium deposits from 1.2- to 1.4-folds by up-regulating the expression of runt-related transcription factor-2 (Runx-2) and bone morphogenetic protein-2 (BMP-2). Meanwhile, HETF suppressed the adipogenesis of MSCs by reducing the formation of adipocyte-like cells and accumulation of fat droplets by down-regulating the expression of peroxisome proliferator-activated receptor-γ (PPAR-γ). The above biological activities of HETF were mainly through estrogen receptor-mediated pathway, which were blocked by estrogen receptor antagonist, ICI 182,780. CONCLUSION: HETF could regulate Runx-2-mediated osteogenesis and PPAR-γ-mediated adipogenesis in MSCs and thus exhibit beneficial effects to bone health, which suggests a new strategy for treating patients with osteoporosis and obesity.

Microfluidic Platform for Studying Chemotaxis of Adhesive Cells Revealed a Gradient-Dependent Migration and Acceleration of Cancer Stem Cells.

Recent studies reveal that solid tumors consist of heterogeneous cells with distinct phenotypes and functions. However, it is unclear how different subtypes of cancer cells migrate under chemotaxis. Here, we developed a microfluidic device capable of generating multiple stable gradients, culturing cells on-chip, and monitoring single cell migratory behavior. The microfluidic platform was used to study gradient-induced chemotaxis of lung cancer stem cell (LCSC) and differentiated LCSC (dLCSC) in real time. Our results showed the dynamic and differential response of both LCSC and dLCSC to chemotaxis, which was regulated by the ß-catenin dependent Wnt signaling pathway. The microfluidic analysis showed that LCSC and dLCSC from the same origin behaved differently in the same external stimuli, suggesting the importance of cancer cell heterogeneity. We also observed for the first time the acceleration of both LCSC and dLCSC during chemotaxis caused by increasing local concentration in different gradients, which could only be realized through the microfluidic approach. The capability to analyze single cell chemotaxis under spatially controlled conditions provides a novel analytical platform for the study of cellular microenvironments and cancer cell metastasis.

On-site formation of emulsions by controlled air plugs.

Air plugs are usually undesirable in microfluidic systems because of their detrimental effect on the system's stability and integrity. By controlling the wetting properties as well as the topographical geometry of the microchannel, it is reported herein that air plugs can be generated in pre-defined locations to function as a unique valve, allowing for the on-site formation of various emulsions including single-component droplets, composite droplets with droplet-to-droplet concentration gradient, blood droplets, paired droplets, as well as bubble arrays without the need for precious flow control, a difficult task with conventional droplet microfluidics. Moreover, the self-generated air valve can be readily deactivated (turned off) by the introduction of an oil phase, allowing for the on-demand release of as-formed droplets for downstream applications. It is proposed that the simple, yet versatile nature of this technique can act as an important method for droplet microfluidics and, in particular, is ideal for the development of affordable lab-on-a-chip systems without suffering from scalability and manufacturing challenges that typically confound the conventional droplet microfluidics.