Application of Cell Painting for chemical hazard evaluation in support of screening-level chemical assessments.

'Cell Painting' is an imaging-based high-throughput phenotypic profiling (HTPP) method in which cultured cells are fluorescently labeled to visualize subcellular structures (i.e., nucleus, nucleoli, endoplasmic reticulum, cytoskeleton, Golgi apparatus / plasma membrane and mitochondria) and to quantify morphological changes in response to chemicals or other perturbagens. HTPP is a high-throughput and cost-effective bioactivity screening method that detects effects associated with many different molecular mechanisms in an untargeted manner, enabling rapid in vitro hazard assessment for thousands of chemicals. Here, 1201 chemicals from the ToxCast library were screened in concentration-response up to ∼100 µM in human U-2 OS cells using HTPP. A phenotype altering concentration (PAC) was estimated for chemicals active in the tested range. PACs tended to be higher than lower bound potency values estimated from a broad collection of targeted high-throughput assays, but lower than the threshold for cytotoxicity. In vitro to in vivo extrapolation (IVIVE) was used to estimate administered equivalent doses (AEDs) based on PACs for comparison to human exposure predictions. AEDs for 18/412 chemicals overlapped with predicted human exposures. Phenotypic profile information was also leveraged to identify putative mechanisms of action and group chemicals. Of 58 known nuclear receptor modulators, only glucocorticoids and retinoids produced characteristic profiles; and both receptor types are expressed in U-2 OS cells. Thirteen chemicals with profile similarity to glucocorticoids were tested in a secondary screen and one chemical, pyrene, was confirmed by an orthogonal gene expression assay as a novel putative GR modulating chemical. Most active chemicals demonstrated profiles not associated with a known mechanism-of-action. However, many structurally related chemicals produced similar profiles, with exceptions such as diniconazole, whose profile differed from other active conazoles. Overall, the present study demonstrates how HTPP can be applied in screening-level chemical assessments through a series of examples and brief case studies.

Evaluating structure-based activity in a high-throughput assay for steroid biosynthesis.

Data from a high-throughput human adrenocortical carcinoma assay (HT-H295R) for steroid hormone biosynthesis are available for >2000 chemicals in single concentration and 654 chemicals in multi-concentration (mc). Previously, a metric describing the effect size of a chemical on the biosynthesis of 11 hormones was derived using mc data referred to as the maximum mean Mahalanobis distance (maxmMd). However, mc HT-H295R assay data remain unavailable for many chemicals. This work leverages existing HT-H295R assay data by constructing structure-activity relationships to make predictions for data-poor chemicals, including: (1) identification of individual structural descriptors, known as ToxPrint chemotypes, associated with increased odds of affecting estrogen or androgen synthesis; (2) a random forest (RF) classifier using physicochemical property descriptors to predict HT-H295R maxmMd binary (positive or negative) outcomes; and, (3) a local approach to predict maxmMd binary outcomes using nearest neighbors (NNs) based on two types of chemical fingerprints (chemotype or Morgan). Individual chemotypes demonstrated high specificity (85-98%) for modulators of estrogen and androgen synthesis but with low sensitivity. The best RF model for maxmMd classification included 13 predicted physicochemical descriptors, yielding a balanced accuracy (BA) of 71% with only modest improvement when hundreds of structural features were added. The best two NN models for binary maxmMd prediction demonstrated BAs of 85 and 81% using chemotype and Morgan fingerprints, respectively. Using an external test set of 6302 chemicals (lacking HT-H295R data), 1241 were identified as putative estrogen and androgen modulators. Combined results across the three classification models (global RF model and two local NN models) predict that 1033 of the 6302 chemicals would be more likely to affect HT-H295R bioactivity. Together, these in silico approaches can efficiently prioritize thousands of untested chemicals for screening to further evaluate their effects on steroid biosynthesis.

Spatial-heterodyne spectrometer for transmission-Raman observations.

A new transmission Raman spectrometer has been developed using a spatial heterodyne spectrometer (SHS), taking advantage of the high etendue inherent in this class of spectrometer to maximize the light collected from the target. The system has been tested against paracetamol tablet samples. The instrument has been shown to accept light from 0.05 mm up to a 3 mm core diameter fibre bundle with a numerical aperture of 0.22, whilst no degradation in resolution is observed.

Scoping review to identify potential non-antimicrobial interventions to mitigate antimicrobial resistance in commensal enteric bacteria in North American cattle production systems.

A scoping review was conducted to identify modifiable non-antimicrobial factors to reduce the occurrence of antimicrobial resistance in cattle populations. Searches were developed to retrieve peer-reviewed published studies in animal, human and in vitro microbial populations. Citations were retained when modifiable non-antimicrobial factors or interventions potentially associated with antimicrobial resistance were described. Studies described resistance in five bacterial genera, species or types, and 40 antimicrobials. Modifiable non-antimicrobial factors or interventions ranged widely in type, and the depth of evidence in animal populations was shallow. Specific associations between a factor or intervention with antimicrobial resistance in a population (e.g. associations between organic systems and tetracycline susceptibility in E. coli from cattle) were reported in a maximum of three studies. The identified non-antimicrobial factors or interventions were classified into 16 themes. Most reported associations between the non-antimicrobial modifiable factors or interventions and antimicrobial resistance were not statistically significant (P > 0·05 and a confidence interval including 1), but when significant, the results were not consistent in direction (increase or decrease in antimicrobial resistance) or magnitude. Research is needed to better understand the impacts of promising modifiable factors or interventions on the occurrence of antimicrobial resistance before any recommendations can be offered or adopted.

Genetic Susceptibility to Rhodococcus equi.

Rhodococcus equi pneumonia is a major cause of morbidity and mortality in neonatal foals. Much effort has been made to identify preventative measures and new treatments for R. equi with limited success. With a growing focus in the medical community on understanding the genetic basis of disease susceptibility, investigators have begun to evaluate the interaction of the genetics of the foal with R. equi. This review describes past efforts to understand the genetic basis underlying R. equi susceptibility and tolerance. It also highlights the genetic technology available to study horses and describes the use of this technology in investigating R. equi. This review provides readers with a foundational understanding of candidate gene approaches, single nucleotide polymorphism-based, and copy number variant-based genome-wide association studies, and next generation sequencing (both DNA and RNA).

Fabry-Pérot optical filter assembly: a candidate for the Mie/ Rayleigh separator in EarthCARE.

This paper describes the development, characterisation and environmental testing of a high performance state-of-the-art optical filter for separating the Mie and Rayleigh backscatter signals for the ATLID LIDAR of the EarthCARE mission. The filter assembly utilises capacitance stabilised Fabry-Pérot étalon technology to provide high resolution optical filtering of the LIDAR signal, efficiently separating the two critical backscatter components from the atmosphere. The paper describes the development of the filter assembly and its subsequent performance testing. The filter has demonstrated state-of-the-art optical performance. Additionally, one of the étalons has been taken through complete environmental testing for space applications.

Microstructured fibers for broadband wavefront filtering in the mid-IR.

The European Space Agency's space-based Darwin mission aims to directly detect extrasolar Earth-like planets using nulling interferometry. However, in order to accomplish this using current optical technology, the interferometer input beams must be filtered to remove local wavefront errors. Although short lengths of single-mode fiber are ideal wavefront filters, Darwin's operating wavelength range of 4 - 20 microm presents real challenges for optical fiber technology. In addition to the fact that step-index fibers only offer acceptable coupling efficiency over about one octave of optical bandwidth, very few suitable materials are transparent within this wavelength range. Microstructured optical fibers offer two unique properties that hold great promise for this application; they can be made from a single-material and offer endlessly single-mode guidance. Here we explore the advantages of using a microstructured fiber as a broadband wavefront filter for 4 - 20 microm.

Nitric oxide stabilizes the Mo(V) oxidation state of dimethyl sulfoxide reductase from Rhodobacter sphaeroides without inhibiting enzyme activity.

Dimethyl sulfoxide reductase from Rhodobacter sphaeroides is isolated in an oxidized, Mo(VI) containing form. Both nitric oxide and reduced ascorbate carried out a one electron reduction of the enzyme with formation of stoichiometric amounts of EPR active Mo(V). Nitric oxide also caused a one electron oxidation of reduced, Mo(IV) enzyme. Mo(V) formation was accompanied by appearance of absorbance peaks at 387 and 528 nm. Neither nitric oxide nor ascorbate inhibited the enzyme nor did either compound support enzyme turnover. Both nitrite plus ascorbate and nitroxyl anion (NO-) induced a previously reported rhombic EPR signal (g1 = 1.994, g2 = 1.982, g3 = 1.966) which exhibited superhyperfine coupling to an exchangeable proton (A1 = 1.25 mT, A2 = 0.85 mT, and A3 = 1.0 mT). On the other hand, NO(g) induced an axial signal with g perpendicular = 1.982 and g parallel = 1.961 in which there is no evidence of superhyperfine coupling. Thus, ascorbate, nitric oxide, and nitric oxide donors induce and stabilize Mo(V) formation in dimethyl sulfoxide reductase without inhibiting enzyme activity. The resemblance between NO and the natural N-oxide substrates of this enzyme suggest that the Mo(V)-NO complex may be a transition state analog of the enzyme-substrate complex.

Pulmonary hypoplasia associated with reduced thoracic space in mice with disproportionate micromelia (DMM).

BACKGROUND: Fetal mice homozygous for the Disproportionate micromelia (Dmm) gene were studied as a model for pulmonary hypoplasia in chondrodystrophy. METHODS: Wet weight, dry weight, and biochemical content were determined in excised whole lungs, terminal sac morphology and presence of multilamellar bodies were determined by electron microscopy, and volume of the thoracic space was estimated from paraffin casts. Lung development of the mutant was further assessed in whole organ culture. RESULTS. Compared with normal littermates, the mutant showed a significant decrease (28%) in lung wet weight without showing altered lung dry weight or tissue content of DNA and protein. The terminal sacs of lungs fixed by intratracheal instillation were significantly smaller than normal. However, the lungs appeared to have undergone maturation on schedule since the surfactant precursors, multilamellar bodies, were observed and normal tissue-levels of phospholipid were detected. The volume of the mutant's thorax was markedly reduced. Finally, the mutant's lungs when removed from the fetus prior to the onset of thoracic dystrophy (day 15) and cultured for three days demonstrated that, without the confining influence of a reduced thoracic space, they are capable of development comparable to normal. CONCLUSIONS: These findings support the hypothesis that the Dmm mutant can be further studied as a model for human pulmonary hypoplasia associated with chondrodystrophy, and that the relationship between the reduced thorax and the lung disorder is cause-and-effect.