Visible blue light inhibits infection and replication of SARS-CoV-2 at doses that are well-tolerated by human respiratory tissue.

The delivery of safe, visible wavelengths of light can be an effective, pathogen-agnostic, countermeasure that would expand the current portfolio of SARS-CoV-2 intervention strategies beyond the conventional approaches of vaccine, antibody, and antiviral therapeutics. Employing custom biological light units, that incorporate optically engineered light-emitting diode (LED) arrays, we harnessed monochromatic wavelengths of light for uniform delivery across biological surfaces. We demonstrated that primary 3D human tracheal/bronchial-derived epithelial tissues tolerated high doses of a narrow spectral band of visible light centered at a peak wavelength of 425 nm. We extended these studies to Vero E6 cells to understand how light may influence the viability of a mammalian cell line conventionally used for assaying SARS-CoV-2. The exposure of single-cell monolayers of Vero E6 cells to similar doses of 425 nm blue light resulted in viabilities that were dependent on dose and cell density. Doses of 425 nm blue light that are well-tolerated by Vero E6 cells also inhibited infection and replication of cell-associated SARS-CoV-2 by > 99% 24 h post-infection after a single five-minute light exposure. Moreover, the 425 nm blue light inactivated cell-free betacoronaviruses including SARS-CoV-1, MERS-CoV, and SARS-CoV-2 up to 99.99% in a dose-dependent manner. Importantly, clinically applicable doses of 425 nm blue light dramatically inhibited SARS-CoV-2 infection and replication in primary human 3D tracheal/bronchial tissue. Safe doses of visible light should be considered part of the strategic portfolio for the development of SARS-CoV-2 therapeutic countermeasures to mitigate coronavirus disease 2019 (COVID-19).

Infrared spectroscopy detects changes in an amphibian cell line induced by fungicides: Comparison of single and mixture effects.

Amphibians are regarded as sensitive sentinels of environmental pollution due to their permeable skin and complex life cycle, which usually involves reproduction and development in the aquatic environment. Fungicides are widely applied agrochemicals and have been associated with developmental defects in amphibians; thus, it is important to determine chronic effects of environmentally-relevant concentrations of such contaminants in target cells. Infrared (IR) spectroscopy has been employed to signature the biological effects of environmental contaminants through extracting key features in IR spectra with chemometric methods. Herein, the Xenopus laevis (A6) cell line was exposed to low concentrations of carbendazim (a benzimidazole fungicide) or flusilazole (a triazole fungicide) either singly or as a binary mixture. Cells were then examined using attenuated total reflection Fourier-transform IR (ATR-FTIR) spectroscopy coupled with multivariate analysis. Results indicate significant changes in the IR spectra of cells induced by both agents at all concentrations following single exposures, primarily in regions associated with protein and phospholipids. Distinct differences were apparent in the IR spectra of cells exposed to carbendazim and those exposed to flusilazole, suggesting different mechanisms of action. Exposure to binary mixtures of carbendazim and flusilazole also induced significant spectral alterations, again in regions associated with phospholipids and proteins, but also in regions associated with DNA and carbohydrates. Overall these findings demonstrate that IR spectroscopy is a sensitive technique for examining the effects of environmentally-relevant levels of fungicides at the cellular level. The combination of IR spectroscopy with the A6 cell line could serve as a useful model to identify agents that might threaten amphibian health in a rapid and high throughput manner.

Biospectroscopy reveals the effect of varying water quality on tadpole tissues of the common frog (Rana temporaria).

Amphibians are undergoing large population declines in many regions around the world. As environmental pollution from both agricultural and urban sources has been implicated in such declines, there is a need for a biomonitoring approach to study potential impacts on this vulnerable class of organism. This study assessed the use of infrared (IR) spectroscopy as a tool to detect changes in several tissues (liver, muscle, kidney, heart and skin) of late-stage common frog (Rana temporaria) tadpoles collected from ponds with differing water quality. Small differences in spectral signatures were revealed between a rural agricultural pond and an urban pond receiving wastewater and landfill run-off; these were limited to the liver and heart, although large differences in body size were apparent, surprisingly with tadpoles from the urban site larger than those from the rural site. Large differences in liver spectra were found between tadpoles from the pesticide and nutrient impacted pond compared to the rural agricultural pond, particularly in regions associated with lipids. Liver mass and hepatosomatic indices were found to be significantly increased in tadpoles from the site impacted by pesticides and trace organic chemicals, suggestive of exposure to environmental contamination. Significant alterations were also found in muscle tissue between tadpoles from these two ponds in regions associated with glycogen, potentially indicative of a stress response. This study highlights the use of IR spectroscopy, a low-cost, rapid and reagent-free technique in the biomonitoring of a class of organisms susceptible to environmental degradation.

Classification of agents using Syrian hamster embryo (SHE) cell transformation assay (CTA) with ATR-FTIR spectroscopy and multivariate analysis.

The Syrian hamster embryo (SHE) cell transformation assay (pH 6.7) has a reported sensitivity of 87% and specificity of 83%, and an overall concordance of 85% with in vivo rodent bioassay data. To date, the SHE assay is the only in vitro assay that exhibits multistage carcinogenicity. The assay uses morphological transformation, the first stage towards neoplasm, as an endpoint to predict the carcinogenic potential of a test agent. However, scoring of morphologically transformed SHE cells is subjective. We treated SHE cells grown on low-E reflective slides with 2,6-diaminotoluene, N-nitroso-N-ethylnitroguanidine, N-nitroso-N-methylurea, N-nitroso-N-ethylurea, EDTA, dimethyl sulphoxide (DMSO; vehicle control), methyl methanesulfonate, benzo[e]pyrene, mitomycin C, ethyl methanesulfonate, ampicillin or five different concentrations of benzo[a]pyrene. Macroscopically visible SHE colonies were located on the slides and interrogated using attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy acquiring five spectra per colony. The acquired IR data were analysed using Fisher's linear discriminant analysis (LDA) followed by principal component analysis (PCA)-LDA cluster vectors to extract major and minor discriminating wavenumbers for each treatment class. Each test agent vs. DMSO and treatment-induced transformed cells vs. corresponding non-transformed were classified by a unique combination of major and minor discriminating wavenumbers. Alterations associated with Amide I, Amide II, lipids and nucleic acids appear to be important in segregation of classes. Our findings suggest that a biophysical approach of ATR-FTIR spectroscopy with multivariate analysis could facilitate a more objective interrogation of SHE cells towards scoring for transformation and ultimately employing the assay for risk assessment of test agents.

Feather corticosterone content in predatory birds in relation to body condition and hepatic metal concentration.

This study investigated the feasibility of measuring corticosterone in feathers from cryo-archived raptor specimens, in order to provide a retrospective assessment of the activity of the stress axis in relation to contaminant burden. Feather samples were taken from sparrowhawk Accipiter nisus, kestrel Falco tinnunculus, buzzard Buteo buteo, barn owl Tyto alba, and tawny owl Strix aluco and the variation in feather CORT concentrations with respect to species, age, sex, feather position, and body condition was assessed. In sparrowhawks only, variation in feather CORT content was compared with hepatic metal concentrations. For individuals, CORT concentration (pgmm(-1)) in adjacent primary flight feathers (P5 and P6), and left and right wing primaries (P5), was statistically indistinguishable. The lowest concentrations of CORT were found in sparrowhawk feathers and CORT concentrations did not vary systematically with age or sex for any species. Significant relationships between feather CORT content and condition were observed in only tawny owl and kestrel. In sparrowhawks, feather CORT concentration was found to be positively related to the hepatic concentrations of five metals (Cd, Mn, Co, Cu, Mo) and the metalloid As. There was also a negative relationship between measures of condition and total hepatic metal concentration in males. The results suggest that some factors affecting CORT uptake by feathers remain to be resolved but feather CORT content from archived specimens has the potential to provide a simple effects biomarker for exposure to environmental contaminants.

Using Fourier transform IR spectroscopy to analyze biological materials.

IR spectroscopy is an excellent method for biological analyses. It enables the nonperturbative, label-free extraction of biochemical information and images toward diagnosis and the assessment of cell functionality. Although not strictly microscopy in the conventional sense, it allows the construction of images of tissue or cell architecture by the passing of spectral data through a variety of computational algorithms. Because such images are constructed from fingerprint spectra, the notion is that they can be an objective reflection of the underlying health status of the analyzed sample. One of the major difficulties in the field has been determining a consensus on spectral pre-processing and data analysis. This manuscript brings together as coauthors some of the leaders in this field to allow the standardization of methods and procedures for adapting a multistage approach to a methodology that can be applied to a variety of cell biological questions or used within a clinical setting for disease screening or diagnosis. We describe a protocol for collecting IR spectra and images from biological samples (e.g., fixed cytology and tissue sections, live cells or biofluids) that assesses the instrumental options available, appropriate sample preparation, different sampling modes as well as important advances in spectral data acquisition. After acquisition, data processing consists of a sequence of steps including quality control, spectral pre-processing, feature extraction and classification of the supervised or unsupervised type. A typical experiment can be completed and analyzed within hours. Example results are presented on the use of IR spectra combined with multivariate data processing.

Mid-infrared spectroscopic assessment of nanotoxicity in gram-negative vs. gram-positive bacteria.

Nanoparticles appear to induce toxic effects through a variety of mechanisms including generation of reactive oxygen species (ROS), physical contact with the cell membrane and indirect catalysis due to remnants from manufacture. The development and subsequent increasing usage of nanomaterials has highlighted a growing need to characterize and assess the toxicity of nanoparticles, particularly those that may have detrimental health effects such as carbon-based nanomaterials (CBNs). Due to interactions of nanoparticles with some reagents, many traditional toxicity tests are unsuitable for use with CBNs. Infrared (IR) spectroscopy is a non-destructive, high throughput technique, which is unhindered by such problems. We explored the application of IR spectroscopy to investigate the effects of CBNs on Gram-negative (Pseudomonas fluorescens) and Gram-positive (Mycobacterium vanbaalenii PYR-1) bacteria. Two types of IR spectroscopy were compared: attenuated total reflection Fourier-transform infrared (ATR-FTIR) and synchrotron radiation-based FTIR (SR-FTIR) spectroscopy. This showed that Gram-positive and Gram-negative bacteria exhibit differing alterations when exposed to CBNs. Gram-positive bacteria appear more resistant to these agents and this may be due to the protection afforded by their more sturdy cell wall. Markers of exposure also vary according to Gram status; Amide II was consistently altered in Gram-negative bacteria and carbohydrate altered in Gram-positive bacteria. ATR-FTIR and SR-FTIR spectroscopy could both be applied to extract biochemical alterations induced by each CBN that were consistent across the two bacterial species; these may represent potential biomarkers of nanoparticle-induced alterations. Vibrational spectroscopy approaches may provide a novel means of fingerprinting the effects of CBNs in target cells.

Incorporation of deuterium oxide in MCF-7 cells to shed further mechanistic insights into benzo[a]pyrene-induced low-dose effects discriminated by ATR-FTIR spectroscopy.

This study evaluated the potential of deuteration to enhance the mechanistic information obtainable by biospectroscopy techniques in biological-cell models. These techniques were previously demonstrated to identify low-dose effects (≤nM) induced by test agents; this is of critical interest in terms of developing novel approaches to monitor environmentally-induced cell alterations. Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy was coupled with multivariate analysis to characterize a low-dose (10(-10) M) compared to a high-dose (10(-6) M) exposure of benzo[a]pyrene (B[a]P) in oestrogen-responsive MCF-7 cells; these results were used as a positive control for spectroscopic detection of B[a]P-induced effects. Deuterium oxide (D2O) was then applied as part of a fixative solution and/or at low levels incorporated into growth medium prior to ATR-FTIR spectrochemical analysis. The application of D2O as an alternative solvent in spectroscopy is widespread, but D2O has never before been applied to biospectroscopic analysis of in vitro toxicology assays. This allowed comparison between deuterated- and typically-derived IR spectra, facilitating significant insights into the effects of deuteration, and suggested that the addition of D2O to biospectroscopy assays could improve understanding of low-dose effects.