To study the toxicity of nanoparticles under relevant conditions, it is critical to disperse reproducibly in different agglomeration states aqueous solutions compatible with cell-based assays. Here, we gold, silver, cerium oxide, and positively-charged polystyrene cell culture media, using timing between mixing steps control agglomerate size otherwise identical media. These protein-stabilized dispersions are generally stable for at least two days, mean sizes ∼23 nm silver ranging from...

A simple yet versatile method was developed to prepare a low-density polymerization initiator gradient, which combined with surface-initiated atom transfer radical (ATRP) produce well-defined poly(2-hydroxyethyl methacrylate) (HEMA) gradient substrate. smooth variation in film thickness measured across the ranging from 20 Å over 80 Å, but we observed nonmonotonic water contact angle. Fits of X-ray reflectivity profiles suggested that at low graft density end, polymer chain structure...

Here we describe the use of electrospray differential mobility analysis (ES-DMA), also known as gas-phase electrophoretic molecular (GEMMA), a method for measuring low-order soluble aggregates proteins in solution. We demonstrate proof concept with IgG antibodies. In ES-DMA, aqueous solutions antibody protein are electrosprayed and various aerosolized species separated according to their using analyzer. this way, complete size distributions present from 3 250 nm can be obtained current set...

The analysis of fluorescence microscopy cells often requires the determination cell edges. This is typically done using segmentation techniques that separate objects in an image from surrounding background. study compares results nine different applied to two lines and five sets imaging conditions. Significant variability was observed due solely differences conditions or applications algorithms. We quantified compared with a novel bivariate similarity index metric evaluates degree...

An important consideration in developing standards and regulations that govern the production use of commercial nanoscale materials is development robust reliable measurements to monitor potential adverse biological effects such products. These typically require cell-based other assays provide an assessment risks associated with nanomaterial interest. In this perspective, we describe cause-and-effect (C&E) analysis design robust, high quality test nanoparticle-related cytotoxicity. C&E assay...

The increased use and incorporation of engineered nanoparticles (ENPs) in consumer products requires a robust assessment their potential environmental implications. However, lack standardized methods for nanotoxicity testing has yielded results that are sometimes contradictory. Standard ecotoxicity assays may work appropriately some ENPs with minimal modification but produce artifactual others. Therefore, understanding the robustness range is critical. In this study, we evaluated performance...

Development of reliable cell-based nanotoxicology assays is important for evaluation potentially hazardous engineered nanomaterials. Challenges to producing a assay protocol include working with nanoparticle dispersions and living cell lines, the potential nano-related interference effects. Here we demonstrate use 96-well plate design several measurement controls an interlaboratory comparison study involving five laboratories characterize robustness nanocytotoxicity MTS viability based on...

Thin films of the extracellular matrix protein, collagen, were prepared by adsorbing native or heat-denatured type I collagen onto hexadecanethiol self-assembled monolayers. The resulting characterized atomic force microscopy, ellipsometry, and light microscopy. Denatured formed a topographically smooth ∼3.6 nm thick film, consistent with an adsorbed protein monolayer. In contrast, thin film consisted supramolecular fibrils. density large fibrils could be varied changing concentration in...

One primary challenge in nanotoxicology studies is the lack of well-characterised nanoparticle reference materials which could be used as positive or negative controls. The National Institute Standards and Technology (NIST) has developed three gold (AuNP) (10, 30 60 nm). genotoxicity these nanoparticles was tested using HepG2 cells calf-thymus DNA. DNA damage assessed based on specific sensitive measurement four oxidatively-modified lesions (8-hydroxy-2´-deoxyguanosine,...

We report the development of a novel cationic dendron (TAG1-PCD) and positively charged gold nanoparticle-dendron conjugate (PCD-AuNP). TAG1-PCD was designed by considering reactivity, hydrophilicity, nature that is required to yield stable in aqueous media. The PCD-AuNPs, nominally 10 nm size, were synthesized reduction chloroauric acid presence TAG1-PCD. physicochemical properties PCD-AuNPs characterized dynamic light scattering, transmission electron microscopy, UV-vis absorbance, X-ray...

Increasing production and use of engineered nanomaterials (ENMs) has generated widespread interest in measuring their environmental human health effects.

For environmental studies assessing uptake of orally ingested engineered nanoparticles (ENPs), a key step in ensuring accurate quantification ENPs is efficient separation the organism from that are either nonspecifically adsorbed to and/or suspended dispersion following exposure. Here, we measure 30 and 60 nm gold (AuNPs) by nematode, Caenorhabditis elegans, using sucrose density gradient centrifugation protocol remove noningested AuNPs. Both conventional inductively coupled plasma mass...

Abstract The nematode Caenorhabditis elegans is used extensively in molecular, toxicological and genetics research. However, standardized methods for counting nematodes liquid culture do not exist despite the wide use of need accurate measurements. Herein, we provide a simple affordable protocol developed to maximize count accuracy minimize variability culture. Sources process were identified tested 14 separate experiments. Three variables resulted significant effects on count: shaking...

The molecular structure of human liver fructose-1,6-bisphosphatase complexed with AMP was determined by x-ray diffraction using replacement, starting from the pig kidney enzyme complex. Of 34 amino acid residues which differ between these two sequences, only one interacts AMP; Met30 in is Leu30 liver. From this analysis, six sites side chains are contact AMP, Ala24, Leu30, Thr31, Tyr113, Arg140, and Met177, were mutated polymerase chain reaction. wild-type mutant forms expressed Escherichia...

Tenascin-C (TN-C) is an extracellular matrix (ECM) protein expressed within remodeling systemic and pulmonary arteries (PAs), where it supports vascular smooth muscle cell (SMC) proliferation. Previously, we showed that A10 SMCs cultivated on native type I collagen possess a spindle-shaped morphology do not express TN-C, whereas those denatured well-defined F-actin stress fiber network, spread morphology, they TN-C. To determine whether changes in cytoskeletal architecture control were...

To accurately interpret the data from fluorescent proteins as reporters of gene activation within living cells, it is important to understand kinetics degradation reporter proteins. We examined over a large number (>1,000) single, cells clonal population NIH3T3 fibroblasts that were stably transfected with destabilized, enhanced green protein (eGFP) driven by tenascin-C promoter. Data collection and quantification fluorescence statistically significant individual long times (14 h) automated...

The cell therapy industry has identified the inability to reliably characterize cells as possibly its greatest challenge and called for standards reference materials provide assurance measurements of properties. challenges in characterization products can be largely addressed with systematic approaches assessing sources uncertainty improving confidence key measurements. This article presents many strategies that used ensure measurement discusses them terms how they applied products....

The current push for rigor and reproducibility is driven by a desire confidence in research results. Here, we suggest framework systematic process, based on consensus principles of measurement science, to guide researchers reviewers assessing, documenting, mitigating the sources uncertainty study. All study results have associated ambiguities that are not always clarified simply establishing reproducibility. By explicitly considering uncertainty, noting aspects experimental system difficult...

COVID-19 is an ongoing, global pandemic caused by the novel, highly infectious SARS-CoV-2 virus. Efforts to mitigate effects of SARS-CoV-2, such as mass vaccination and development monoclonal therapeutics, require precise measurements correlative, functional neutralizing antibodies that block virus infection. The rapid, safe, easy-to-use neutralization assays essential for faster diagnosis treatment. Here, we developed a vesicular stomatitis (VSV)-based assay with two readout methods,...

Phospholipase C (PLC)-β 1 and PLC-β 2 are regulated by the G q family of heterotrimeric proteins contain C2 domains. These domains Ca 2+ -binding modules that serve as membrane-attachment motifs in a number signal transduction proteins. To determine role play function, we measured binding isolated to membrane bilayers. We found, unexpectedly, these do not bind membranes but they associate strongly specifically activated [guanosine 5′-[γ-thio]triphosphate (GTP[γS])-bound] Gα subunits. The...