nanoDSF: In vitro Label-Free Method to Monitor Picornavirus Uncoating and Test Compounds Affecting Particle Stability

Abstract Background Despite the increased morbidity of ulcerative colitis (UC) in the developing countries, available treatments remain unsatisfactory. Therefore, it is urgent to discover more effective therapeutic strategies. Pulsatilla chinensis was widely used for the treatment of inflamed intestinal diseases including UC for thousands of years in China. Anemoside B4, the most abundant triterpenoid saponin isolated from P. chinensis, exerts anti-inflammatory and antioxidant effects and may be the most active compounds, which is responsible for the therapeutic effects. However, the mechanism how anemoside B4 executes its biological functions is still elusive. Methods Here, we used the 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-induced colitis rat model to evaluate the therapeutic effect of anemoside B4. Blood samples of colitis rats were collected for hematology analysis. The inflammation-associated factors were investigated by enzyme-linked immunosorbent assay (ELISA). Cell proliferation and apoptosis was determined with EdU cell proliferation assay and TUNEL assay. The proteins regulated by anemoside B4 were identified by label-free quantitative proteomics. The significantly down-regulated proteins were verified by Western blotting analysis. mRNA expression was analyzed by quantitative real-time RT-PCR. Results The results showed that anemoside B4 ameliorated TNBS-induced colitis symptoms, including tissue damage, inflammatory cell infiltration, and pro-inflammatory cytokine production, apoptosis and slowed proliferation in colon. Quantitative proteomic analyses discovered that 56 proteins were significantly altered by anemoside B4 in the TNBS-induced rats. These proteins mainly clustered in tricarboxylic acid (TCA) cycle and respiratory electron transport chain. Among the altered proteins, S100A9 is one of the most significantly down-regulated proteins and associated with NF-κB and MAPK signaling pathways in the pathogenesis of UC. Further experiments revealed that anemoside B4 suppressed the expression of S100A9 and its downstream genes including TLR4 and NF-κB in colon. In vitro, anemoside B4 could inhibit the NF-κB signaling pathway induced by recombinant S100A9 protein in human intestinal epithelial Caco-2 cells. Moreover, anemoside B4 inhibits neutrophils recruitment and activation in colon induced by TNBS. Conclusions Our results demonstrate that anemoside B4 prevents TNBS-induced colitis by inhibiting the NF-κB signaling pathway through deactivating S100A9, suggesting that anemoside B4 is a promising therapeutic candidate for colitis.

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Low-Molecular-Weight Polyethyleneimine Grafted Polythiophene for Efficient siRNA Delivery

BioMed Research International ◽

10.1155/2015/406389 ◽

2015 ◽

Vol 2015 ◽

pp. 1-9 ◽

Cited By ~ 4

Author(s):

Pan He ◽

Kyoji Hagiwara ◽

Hui Chong ◽

Hsiao-hua Yu ◽

Yoshihiro Ito

Keyword(s):

Molecular Weight ◽

Sirna Delivery ◽

Fluorescent Microscopy ◽

Molar Ratio ◽

Low Molecular Weight ◽

Label Free ◽

Endosomal Membrane ◽

Cationic Copolymers ◽

Interfering Rna

Owing to its hydrophilicity, negative charge, small size, and labile degradation by endogenous nucleases, small interfering RNA (siRNA) delivery must be achieved by a carrier system. In this study, cationic copolymers composed of low-molecular-weight polyethylenimine and polythiophenes were synthesized and evaluated as novel self-tracking siRNA delivery vectors. The concept underlying the design of these copolymers is that hydrophobicity and rigidity of polythiophenes should enhance the transport of siRNA across the cell membrane and endosomal membrane. A gel retardation assay showed that the nanosized complexes formed between the copolymers and siRNA were stable even at a molar ratio of 1 : 2. The high cellular uptake (>80%) and localization of the copolymer vectors inside the cells were easily analyzed by tracking the fluorescence of polythiophene using fluorescent microscopy and cytometry. Anin vitroluciferase knockdown (KD) assay in A549-luc cells demonstrated that the siRNA complexes with more hydrophobic copolymers achieved a higher KD efficiency of 52.8% without notable cytotoxicity, indicating protein-specific KD activity rather than solely the cytotoxicity of the materials. Our polythiophene copolymers should serve as novel, efficient, low cell toxicity, and label-free siRNA delivery systems.

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Y-shaped probe for convenient and label-free detection of microRNA-21 in vitro

Analytical Biochemistry ◽

10.1016/j.ab.2016.01.022 ◽

2016 ◽

Vol 499 ◽

pp. 8-14 ◽

Cited By ~ 13

Author(s):

Kui He ◽

Rong Liao ◽

Changqun Cai ◽

Caishuang Liang ◽

Chan Liu ◽

...

Keyword(s):

Label Free ◽

Label Free Detection

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Studies of the interaction of ticagrelor with the P2Y13 receptor and with P2Y13-dependent pro-platelet formation by human megakaryocytes

Thrombosis and Haemostasis ◽

10.1160/th15-10-0829 ◽

2016 ◽

Vol 116 (12) ◽

pp. 1079-1088 ◽

Cited By ~ 9

Author(s):

Anna Björquist ◽

Christian A. Di Buduo ◽

Eti A. Femia ◽

Robert F. Storey ◽

Richard C. Becker ◽

...

Keyword(s):

Platelet Count ◽

Cellular Response ◽

P2y Receptors ◽

Cell System ◽

Experimental Models ◽

Significant Activity ◽

Label Free ◽

Coronary Syndrome ◽

Platelet Formation

SummaryTicagrelor is an antagonist of the platelet P2Y12 receptor for ADP, approved for the prevention of thromboembolic events in patients with acute coronary syndrome. Previous studies showed that ticagrelor has no significant activity versus P1 receptors for adenosine and other known P2Y receptors, with the exception of P2Y13, which was not tested. The P2Y12 antagonist cangrelor has been shown to also inhibit P2Y13 and to decrease the P2Y13-regulated capacity of megakaryocytes to produce pro-platelets. We tested whether or not ticagrelor inhibits P2Y13 signalling and function. The in vitro effects of ticagrelor, its active (TAM) and inactive (TIM) metabolites, cangrelor and the P2Y13 antagonist MRS2211 were tested in two experimental models: 1) a label-free cellular response assay in P2Y13-transfected HEK293 T-REx cells; and 2) pro-platelet formation by human megakaryocytes in culture. Ticagrelor, TAM, cangrelor and MRS2211, but not TIM, inhibited the cellular responses in P2Y13-transfected cells. In contrast, only MRS2211 and cangrelor, confirming previous results, inhibited pro-platelet formation by megakaryocytes in vitro. The platelet count of patients randomised to treatment with ticagrelor in the PLATO trial did not change during treatment and was comparable to those of patients randomised to clopidogrel. In conclusion, ticagrelor and TAM act as P2Y13 antagonists in a transfected cell system in vitro but this does not translate into any impact on pro-platelet formation in vitro or altered platelet count in patients.

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Distinct roles of tumor associated mutations in collective cell migration

Scientific Reports ◽

10.1038/s41598-021-89130-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Rachel M. Lee ◽

Michele I. Vitolo ◽

Wolfgang Losert ◽

Stuart S. Martin

Keyword(s):

Collective Behavior ◽

Metastatic Potential ◽

Breast Epithelial Cell ◽

Collective Cell Migration ◽

Label Free ◽

Collective Migration ◽

Contrast Imaging ◽

Pten Deletion

AbstractRecent evidence suggests that groups of cells are more likely to form clinically dangerous metastatic tumors, emphasizing the importance of understanding mechanisms underlying collective behavior. The emergent collective behavior of migrating cell sheets in vitro has been shown to be disrupted in tumorigenic cells but the connection between this behavior and in vivo tumorigenicity remains unclear. We use particle image velocimetry to measure a multidimensional migration phenotype for genetically defined human breast epithelial cell lines that range in their in vivo behavior from non-tumorigenic to aggressively metastatic. By using cells with controlled mutations, we show that PTEN deletion enhances collective migration, while Ras activation suppresses it, even when combined with PTEN deletion. These opposing effects on collective migration of two mutations that are frequently found in patient tumors could be exploited in the development of novel treatments for metastatic disease. Our methods are based on label-free phase contrast imaging, and thus could easily be applied to patient tumor cells. The short time scales of our approach do not require potentially selective growth, and thus in combination with label-free imaging would allow multidimensional collective migration phenotypes to be utilized in clinical assessments of metastatic potential.

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Development of Bioimpedance Sensors and Measurement System for Biomedical In-Vitro Applications

Current Directions in Biomedical Engineering ◽

10.1515/cdbme-2021-2126 ◽

2021 ◽

Vol 7 (2) ◽

pp. 496-499

Author(s):

Stadler B. Eng. Sebastian ◽

Herbert Plischke ◽

Christian Hanshans

Keyword(s):

Measurement System ◽

Low Cost ◽

Measurement Data ◽

Impedance Measurement ◽

Microtiter Plate ◽

Label Free ◽

Single Chip ◽

Do It Yourself ◽

Store And Forward

Abstract Bioimpedance analysis is a label-free and easy approach to obtain information on cellular barrier integrity and cell viability more broadly. In this work, we introduce a small, low-cost, portable in vitro impedance measurement system for studies where a shadow-free exposure of the cells is a requirement. It can be controlled by a user-friendly web interface and can perform measurements automated and autonomously at short intervals. The system can be integrated into an existing IoT network for remote monitoring and indepth analyses. A single-board computer (SBC) serves as the central unit, to control, analyze, store and forward the measurement data from the single-chip impedance analyzer. Various materials and manufacturing methods were used to produce a purpose-built lid on top of a modified 24-well microtiter plate in a “do it yourself” fashion. Furthermore, three different sensor designs were developed utilizing anodic aluminum oxide (AAO) membranes and gold-plated electrodes. Preliminary tests with potassium chloride (KCl) showed first promising results.

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Redox-Type Label-Free ATP Image Sensor for Highly Sensitive in Vitro Imaging of Extracellular ATP

2021 21st International Conference on Solid-State Sensors, Actuators and Microsystems (Transducers) ◽

10.1109/transducers50396.2021.9495500 ◽

2021 ◽

Author(s):

Hideo Doi ◽

Tomoko Horio ◽

Young-Joon Choi ◽

Kazuhiro Takahashi ◽

Toshihiko Noda ◽

...

Keyword(s):

Image Sensor ◽

Extracellular Atp ◽

Label Free ◽

Highly Sensitive

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Quantifying single-cell secretion in real time using resonant hyperspectral imaging

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1814977115 ◽

2018 ◽

Vol 115 (52) ◽

pp. 13204-13209 ◽

Cited By ~ 11

Author(s):

José Juan-Colás ◽

Ian S. Hitchcock ◽

Mark Coles ◽

Steven Johnson ◽

Thomas F. Krauss

Keyword(s):

Single Cell ◽

Real Time ◽

Protein Secretion ◽

Cell Communication ◽

Label Free ◽

Cell Secretion ◽

Physiological Disorder ◽

Real Time Analysis ◽

Technology Heterogeneity

Cell communication is primarily regulated by secreted proteins, whose inhomogeneous secretion often indicates physiological disorder. Parallel monitoring of innate protein-secretion kinetics from individual cells is thus crucial to unravel systemic malfunctions. Here, we report a label-free, high-throughput method for parallel, in vitro, and real-time analysis of specific single-cell signaling using hyperspectral photonic crystal resonant technology. Heterogeneity in physiological thrombopoietin expression from individual HepG2 liver cells in response to platelet desialylation was quantified demonstrating how mapping real-time protein secretion can provide a simple, yet powerful approach for studying complex physiological systems regulating protein production at single-cell resolution.

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Volumetric registration of magnetic nanoparticles for optimization of quantitative immunochromatographic assays for detection of small molecules

EPJ Web of Conferences ◽

10.1051/epjconf/201818510006 ◽

2018 ◽

Vol 185 ◽

pp. 10006 ◽

Cited By ~ 2

Author(s):

Natalia V. Guteneva ◽

Sergey L. Znoyko ◽

Alexey V. Orlov ◽

Maxim P. Nikitin ◽

Petr I. Nikitin

Keyword(s):

Magnetic Nanoparticles ◽

Small Molecules ◽

Optical Methods ◽

Magnetic Method ◽

Label Free ◽

Spectral Correlation ◽

Entire Volume ◽

Specificity And Sensitivity ◽

Particle Quantification

Precise quantitative and highly sensitive detection of small molecules (haptens) is highly demanded in medicine, food quality control, in vitro diagnostics, criminalistics, environmental monitoring, etc. In the present work, the magnetic method of particle quantification and the optical methods of spectral correlation and spectral phase interferometry complement each other for optimization of a quantitative assay for measuring concentrations of small molecules. The assay employs magnetic nanoparticles as labels in rapid immunochromatographic format. The approach was demonstrated with fluorescein as a model molecule. The interferometric label-free biosensors were employed for selection of optimal reagents that produced high specificity and sensitivity. The method of magnetic particle quantification counted the magnetic labels over the entire volume of the immunochromatographic membrane to provide their distribution along the test strip. Such distribution was used for optimization of such parameters as concentrations of the used reagents and of antibody immobilized on the labels, amount of the labels and conjugates of haptens with protein carriers to realize the advanced quantitative immunochromatographic assay.

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Multiscale molecular profiling of pathological bone resolves sexually dimorphic control of extracellular matrix composition

Disease Models & Mechanisms ◽

10.1242/dmm.048116 ◽

2021 ◽

Vol 14 (3) ◽

pp. dmm048116 ◽

Cited By ~ 1

Author(s):

Aikta Sharma ◽

Alice Goring ◽

Peter B. Johnson ◽

Roger J. H. Emery ◽

Eric Hesse ◽

...

Keyword(s):

Extracellular Matrix ◽

Ex Vivo ◽

Second Harmonic ◽

Collagen Fibre ◽

Matrix Composition ◽

Label Free ◽

Fibre Number ◽

Backscattered Electron ◽

Matrix Mineralisation

ABSTRACTCollagen assembly during development is essential for successful matrix mineralisation, which determines bone quality and mechanocompetence. However, the biochemical and structural perturbations that drive pathological skeletal collagen configuration remain unclear. Deletion of vascular endothelial growth factor (VEGF; also known as VEGFA) in bone-forming osteoblasts (OBs) induces sex-specific alterations in extracellular matrix (ECM) conformation and mineralisation coupled to vascular changes, which are augmented in males. Whether this phenotypic dimorphism arises as a result of the divergent control of ECM composition and its subsequent arrangement is unknown and is the focus of this study. Herein, we used murine osteocalcin-specific Vegf knockout (OcnVEGFKO) and performed ex vivo multiscale analysis at the tibiofibular junction of both sexes. Label-free and non-destructive polarisation-resolved second-harmonic generation (p-SHG) microscopy revealed a reduction in collagen fibre number in males following the loss of VEGF, complemented by observable defects in matrix organisation by backscattered electron scanning electron microscopy. This was accompanied by localised divergence in collagen orientation, determined by p-SHG anisotropy measurements, as a result of OcnVEGFKO. Raman spectroscopy confirmed that the effect on collagen was linked to molecular dimorphic VEGF effects on collagen-specific proline and hydroxyproline, and collagen intra-strand stability, in addition to matrix carbonation and mineralisation. Vegf deletion in male and female murine OB cultures in vitro further highlighted divergence in genes regulating local ECM structure, including Adamts2, Spp1, Mmp9 and Lama1. Our results demonstrate the utility of macromolecular imaging and spectroscopic modalities for the detection of collagen arrangement and ECM composition in pathological bone. Linking the sex-specific genetic regulators to matrix signatures could be important for treatment of dimorphic bone disorders that clinically manifest in pathological nano- and macro-level disorganisation.This article has an associated First Person interview with the first author of the paper.

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