Are the answers to biological questions obtained via live fluorescence microscopy substantially affected by phototoxicity? Although a single set of standards for assessing phototoxicity cannot exist owing to the breadth of samples and experimental questions associated with biological imaging, we need quantitative, practical assessments and reporting standards to ensure that imaging has a minimal impact on observed biological processes and sample health. Here we discuss the problem of phototoxicity in biology and suggest guidelines to improve its reporting and assessment.
Phototoxicity is a significant constraint for live cell fluorescence microscopy. Excessive excitation light intensities change the homeostasis of the observed cells. Erroneous and misleading conclusions may be the problematic consequence of observing such light-induced pathophysiology. In this study, we assess the effect of blue light, as commonly used for GFP and YFP excitation, on a motile mammalian cell line. Tracking PC3 cells at different light doses and intensities, we show how motility can be used to reliably assess subtle positive and negative effects of illumination. We further show that the effects are a factor of intensity rather than light dose. Mitotic delay was not a sensitive indicator of phototoxicity. For early detection of the effect of blue light, we analysed the expression of genes involved in oxidative stress. This study addresses the need for relatively simple and sensitive methods to establish a dose-response curve for phototoxicity in mammalian cell line models. We conclude with a working model for phototoxicity and recommendations for its assessment.
Heavy metals are the major concern of the modern age. Among the heavy metals, chromium (Cr(VI)) is regarded as a highly toxic heavy metal released largely from leather tanning operations. To remove such high concentrations of Cr(VI), an advanced method is required urgently. Thus, biosorption using biochar, which is an organic material produced from various sources such as walnut shell, can be applied successfully for Cr(VI) abatement. The major objectives of this experiment were the remediation of the Cr(VI) heavy metal using walnut shell biochar and checking of the effect of pH, biochar dosage, Cr level, and shaking time. Remediation of Cr(VI) using walnut shell biochar was proved to be effective and removed the maximum concentration of Cr(VI) up to 93% at pH 5.5, 2 h agitation time, and the biochar amount of 1.1 g L−1 from an aqueous solution. Equilibrium modeling demonstrated that the chemisorption process was involved in adsorption of Cr(VI). The surface of the biochar was porous and provided numerous sites for Cr(VI) attachment, which was also confirmed by the presence of Cr(VI) onto the biochar after adsorption. Hence, the use of walnut shell biochar was highly effective as a sorbent, which could conveniently be applied to small-scale as well as large-scale levels.
Interactions between pairs of membrane-bound receptors can enhance tumour development with implications for targeted therapies for cancer. Here we demonstrate clear heterotypic interaction between CD74 and CD44, which might act in synergy and hence contribute to breast cancer progression. CD74, a type II transmembrane glycoprotein, is a chaperone for MHC class II biosynthesis and a receptor for the MIF. CD44 is the receptor for hyaluronic acid and is a Type I transmembrane protein. Interactions between CD74, MIF and the intra-cytoplasmic domain of CD44 result in activation of ERK1/2 pathway, leading to increased cell proliferation and decreased apoptosis. The level of CD44 in the breast tumor cell lines CAMA-1, MDA-MB-231, MDA-MB-435 and the immortalized normal luminal cell line 226LDM was higher than that of CD74. It was also observed that CD74 and CD44 exhibit significant variation in expression levels across the cells. CD74 and CD44 were observed to accumulate in cytoplasmic compartments, suggesting they associate with each other to facilitate tumour growth and metastasis. Use of a novel and validated colocalisation and image processing approach, coupled with co-immunoprecipitation, confirmed that CD74 and CD44 physically interact, suggesting a possible role in breast tumour growth. This is the first time that CD74 and CD44 colocalization has been quantified in breast cancer cells using a non-invasive and validated bioimaging procedure. Measuring the co-expression levels of CD74 and CD44 could potentially be used as a ‘biomarker signature’ to monitor different stages of breast cancer.
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