A multi-biomarker micronucleus assay using imaging flow cytometry.

Genetic toxicity testing assesses the potential of compounds to cause DNA damage. There are many genetic toxicology screening assays designed to assess the DNA damaging potential of chemicals in early drug development aiding the identification of promising drugs that have low-risk potential for causing genetic damage contributing to cancer risk in humans. Despite this, in vitro tests generate a high number of misleading positives, the consequences of which can lead to unnecessary animal testing and/or the abandonment of promising drug candidates. Understanding chemical Mode of Action (MoA) is vital to identifying the true genotoxic potential of substances and, therefore, the risk translation into the clinic. Here we demonstrate a simple, robust protocol for staining fixed, human-lymphoblast p53 proficient TK6 cells with antibodies against É£H2AX, p53 and pH3S28 along with DRAQ5™ DNA staining that enables analysis of un-lysed cells via microscopy approaches such as imaging flow cytometry. Here, we used the Cytek® Amnis® ImageStream®X Mk II which provides a high-throughput acquisition platform with the sensitivity of flow cytometry and spatial morphological information associated with microscopy. Using the ImageStream manufacturer's software (IDEAS® 6.2), a masking strategy was developed to automatically detect and quantify micronucleus events (MN) and characterise biomarker populations. The gating strategy developed enables the generation of a template capable of automatically batch processing data files quantifying cell-cycle, MN, É£H2AX, p53 and pH3 populations simultaneously. In this way, we demonstrate how a multiplex system enables DNA damage assessment alongside MN identification using un-lysed cells on the imaging flow cytometry platform. As a proof-of-concept, we use the tool chemicals carbendazim and methyl methanesulphonate (MMS) to demonstrate the assay's ability to correctly identify clastogenic or aneugenic MoAs using the biomarker profiles established.

An ovine model for investigation of the microenvironment of the male mammary gland.

The specific biology of the male breast remains relatively unexplored in spite of the increasing global prevalence of male breast cancer. Delineation of the microenvironment of the male breast is restricted by the low availability of human samples and a lack of characterisation of appropriate animal models. Unlike the mouse, the male ovine gland persists postnatally. We suggest that the male ovine mammary gland constitutes a promising adjunctive model for the male breast. In this study, we evaluate the male ovine mammary gland microenvironment, comparing intact and neutered males. Assessment of the glandular histo-anatomy highlights the resemblance of the male gland to that of neonatal female sheep and confirms the presence of rudimentary terminal duct lobular units. Irrespective of neutered status, cell proliferation in epithelial and stromal compartments is similarly low in males, and cell proliferation in epithelial cells and in the intralobular stroma is significantly lower than in pubertal female sheep. Between 42% and 72% of the luminal mammary epithelial cells in the male gland express the androgen receptor and expression is significantly reduced by neutering. Luminal epithelial cells within the intact and neutered male gland also express oestrogen receptor alpha, but minimal progesterone receptor expression is observed. The distribution of leukocytes within the ducts and stroma is similar to the mammary gland of female sheep and females of other species. Both macrophages and T lymphocytes are intercalated in the epithelial bilayer and are more abundant in the intralobular stroma than the interlobular stroma, suggesting that they may have a protective immunological function within the vestigial glandular tissue of the male sheep. Mast cells are also observed within the stroma. These cells cluster near the glandular tissue and are frequently located adjacent to blood vessels. The abundance of mast cells is significantly higher in intact males compared to neutered males, suggesting that hormone signalling may impact mast cell recruitment. In this study, we demonstrate the utility of the male ovine mammary gland as a model for furthering our knowledge of postnatal male mammary biology.

In the Murine and Bovine Maternal Mammary Gland Signal Transducer and Activator of Transcription 3 is Activated in Clusters of Epithelial Cells around the Day of Birth.

Signal transducers and activators of transcription (STAT) proteins regulate mammary development. Here we investigate the expression of phosphorylated STAT3 (pSTAT3) in the mouse and cow around the day of birth. We present localised colocation analysis, applicable to other mammary studies requiring identification of spatially congregated events. We demonstrate that pSTAT3-positive events are multifocally clustered in a non-random and statistically significant fashion. Arginase-1 expressing cells, consistent with macrophages, exhibit distinct clustering within the periparturient mammary gland. These findings represent a new facet of mammary STAT3 biology, and point to the presence of mammary sub-microenvironments.

N-Nitrosodimethylamine investigations in Muta™Mouse define point-of-departure values and demonstrate less-than-additive somatic mutant frequency accumulations.

The N-nitrosamine, N-nitrosodimethylamine (NDMA), is an environmental mutagen and rodent carcinogen. Small levels of NDMA have been identified as an impurity in some commonly used drugs, resulting in several product recalls. In this study, NDMA was evaluated in an OECD TG-488 compliant Muta™Mouse gene mutation assay (28-day oral dosing across seven daily doses of 0.02-4 mg/kg/day) using an integrated design that assessed mutation at the transgenic lacZ locus in various tissues and at the endogenous Pig-a gene-locus, along with micronucleus frequencies in peripheral blood. Liver pathology was determined together with NDMA exposure in blood and liver. The additivity of mutation induction was assessed by including two acute single-dose treatment groups (i.e. 5 and 10 mg/kg dose on Day 1), which represented the same total dose as two of the repeat dose treatment groups. NDMA did not induce statistically significant increases in mean lacZ mutant frequency (MF) in bone marrow, spleen, bladder, or stomach, nor in peripheral blood (Pig-a mutation or micronucleus induction) when tested up to 4 mg/kg/day. There were dose-dependent increases in mean lacZ MF in the liver, lung, and kidney following 28-day repeat dosing or in the liver and kidney after a single dose (10 mg/kg). No observed genotoxic effect levels (NOGEL) were determined for the positive repeat dose-response relationships. Mutagenicity did not exhibit simple additivity in the liver since there was a reduction in MF following NDMA repeat dosing compared with acute dosing for the same total dose. Benchmark dose modelling was used to estimate point of departure doses for NDMA mutagenicity in Muta™Mouse and rank order target organ tissue sensitivity (liver > kidney or lung). The BMD50 value for liver was 0.32 mg/kg/day following repeat dosing (confidence interval 0.21-0.46 mg/kg/day). In addition, liver toxicity was observed at doses of ≥ 1.1 mg/kg/day NDMA and correlated with systemic and target organ exposure. The integration of these results and their implications for risk assessment are discussed.

OPTIMAL: An OPTimized Imaging Mass cytometry AnaLysis framework for benchmarking segmentation and data exploration.

Analysis of imaging mass cytometry (IMC) data and other low-resolution multiplexed tissue imaging technologies is often confounded by poor single-cell segmentation and suboptimal approaches for data visualization and exploration. This can lead to inaccurate identification of cell phenotypes, states, or spatial relationships compared to reference data from single-cell suspension technologies. To this end we have developed the "OPTimized Imaging Mass cytometry AnaLysis (OPTIMAL)" framework to benchmark any approaches for cell segmentation, parameter transformation, batch effect correction, data visualization/clustering, and spatial neighborhood analysis. Using a panel of 27 metal-tagged antibodies recognizing well-characterized phenotypic and functional markers to stain the same Formalin-Fixed Paraffin Embedded (FFPE) human tonsil sample tissue microarray over 12 temporally distinct batches we tested several cell segmentation models, a range of different arcsinh cofactor parameter transformation values, 5 different dimensionality reduction algorithms, and 2 clustering methods. Finally, we assessed the optimal approach for performing neighborhood analysis. We found that single-cell segmentation was improved by the use of an Ilastik-derived probability map but that issues with poor segmentation were only really evident after clustering and cell type/state identification and not always evident when using "classical" bivariate data display techniques. The optimal arcsinh cofactor for parameter transformation was 1 as it maximized the statistical separation between negative and positive signal distributions and a simple Z-score normalization step after arcsinh transformation eliminated batch effects. Of the five different dimensionality reduction approaches tested, PacMap gave the best data structure with FLOWSOM clustering out-performing phenograph in terms of cell type identification. We also found that neighborhood analysis was influenced by the method used for finding neighboring cells with a "disc" pixel expansion outperforming a "bounding box" approach combined with the need for filtering objects based on size and image-edge location. Importantly, OPTIMAL can be used to assess and integrate with any existing approach to IMC data analysis and, as it creates .FCS files from the segmentation output and allows for single-cell exploration to be conducted using a wide variety of accessible software and algorithms familiar to conventional flow cytometrists.

Distinct lung cell signatures define the temporal evolution of diffuse alveolar damage in fatal COVID-19.

BACKGROUND: Lung damage in severe COVID-19 is highly heterogeneous however studies with dedicated spatial distinction of discrete temporal phases of diffuse alveolar damage (DAD) and alternate lung injury patterns are lacking. Existing studies have also not accounted for progressive airspace obliteration in cellularity estimates. We used an imaging mass cytometry (IMC) analysis with an airspace correction step to more accurately identify the cellular immune response that underpins the heterogeneity of severe COVID-19 lung disease. METHODS: Lung tissue was obtained at post-mortem from severe COVID-19 deaths. Pathologist-selected regions of interest (ROIs) were chosen by light microscopy representing the patho-evolutionary spectrum of DAD and alternate disease phenotypes were selected for comparison. Architecturally normal SARS-CoV-2-positive lung tissue and tissue from SARS-CoV-2-negative donors served as controls. ROIs were stained for 40 cellular protein markers and ablated using IMC before segmented cells were classified. Cell populations corrected by ROI airspace and their spatial relationships were compared across lung injury patterns. FINDINGS: Forty patients (32M:8F, age: 22-98), 345 ROIs and >900k single cells were analysed. DAD progression was marked by airspace obliteration and significant increases in mononuclear phagocytes (MnPs), T and B lymphocytes and significant decreases in alveolar epithelial and endothelial cells. Neutrophil populations proved stable overall although several interferon-responding subsets demonstrated expansion. Spatial analysis revealed immune cell interactions occur prior to microscopically appreciable tissue injury. INTERPRETATION: The immunopathogenesis of severe DAD in COVID-19 lung disease is characterised by sustained increases in MnPs and lymphocytes with key interactions occurring even prior to lung injury is established. FUNDING: UK Research and Innovation/Medical Research Council through the UK Coronavirus Immunology Consortium, Barbour Foundation, General Sir John Monash Foundation, Newcastle University, JGW Patterson Foundation, Wellcome Trust.

Ames test study designs for nitrosamine mutagenicity testing: qualitative and quantitative analysis of key assay parameters.

The robust control of genotoxic N-nitrosamine (NA) impurities is an important safety consideration for the pharmaceutical industry, especially considering recent drug product withdrawals. NAs belong to the 'cohort of concern' list of genotoxic impurities (ICH M7) because of the mutagenic and carcinogenic potency of this chemical class. In addition, regulatory concerns exist regarding the capacity of the Ames test to predict the carcinogenic potential of NAs because of historically discordant results. The reasons postulated to explain these discordant data generally point to aspects of Ames test study design. These include vehicle solvent choice, liver S9 species, bacterial strain, compound concentration, and use of pre-incubation versus plate incorporation methods. Many of these concerns have their roots in historical data generated prior to the harmonization of Ames test guidelines. Therefore, we investigated various Ames test assay parameters and used qualitative analysis and quantitative benchmark dose modelling to identify which combinations provided the most sensitive conditions in terms of mutagenic potency. Two alkyl-nitrosamines, N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) were studied. NDMA and NDEA mutagenicity was readily detected in the Ames test and key assay parameters were identified that contributed to assay sensitivity rankings. The pre-incubation method (30-min incubation), appropriate vehicle (water or methanol), and hamster-induced liver S9, alongside Salmonella typhimurium strains TA100 and TA1535 and Escherichia coli strain WP2uvrA(pKM101) provide the most sensitive combination of assay parameters in terms of NDMA and NDEA mutagenic potency in the Ames test. Using these parameters and further quantitative benchmark dose modelling, we show that N-nitrosomethylethylamine (NMEA) is positive in Ames test and therefore should no longer be considered a historically discordant NA. The results presented herein define a sensitive Ames test design that can be deployed for the assessment of NAs to support robust impurity qualifications.

Interpretation of in vitro concentration-response data for risk assessment and regulatory decision-making: Report from the 2022 IWGT quantitative analysis expert working group meeting.

Quantitative risk assessments of chemicals are routinely performed using in vivo data from rodents; however, there is growing recognition that non-animal approaches can be human-relevant alternatives. There is an urgent need to build confidence in non-animal alternatives given the international support to reduce the use of animals in toxicity testing where possible. In order for scientists and risk assessors to prepare for this paradigm shift in toxicity assessment, standardization and consensus on in vitro testing strategies and data interpretation will need to be established. To address this issue, an Expert Working Group (EWG) of the 8th International Workshop on Genotoxicity Testing (IWGT) evaluated the utility of quantitative in vitro genotoxicity concentration-response data for risk assessment. The EWG first evaluated available in vitro methodologies and then examined the variability and maximal response of in vitro tests to estimate biologically relevant values for the critical effect sizes considered adverse or unacceptable. Next, the EWG reviewed the approaches and computational models employed to provide human-relevant dose context to in vitro data. Lastly, the EWG evaluated risk assessment applications for which in vitro data are ready for use and applications where further work is required. The EWG concluded that in vitro genotoxicity concentration-response data can be interpreted in a risk assessment context. However, prior to routine use in regulatory settings, further research will be required to address the remaining uncertainties and limitations.

Label-free cell segmentation of diverse lymphoid tissues in 2D and 3D.

Unlocking and quantifying fundamental biological processes through tissue microscopy requires accurate, in situ segmentation of all cells imaged. Currently, achieving this is complex and requires exogenous fluorescent labels that occupy significant spectral bandwidth, increasing the duration and complexity of imaging experiments while limiting the number of channels remaining to address the study's objectives. We demonstrate that the excitation light reflected during routine confocal microscopy contains sufficient information to achieve accurate, label-free cell segmentation in 2D and 3D. This is achieved using a simple convolutional neural network trained to predict the probability that reflected light pixels belong to either nucleus, cytoskeleton, or background classifications. We demonstrate the approach across diverse lymphoid tissues and provide video tutorials demonstrating deployment in Python and MATLAB or via standalone software for Windows.

Spatial statistics is a comprehensive tool for quantifying cell neighbor relationships and biological processes via tissue image analysis.

Automated microscopy and computational image analysis has transformed cell biology, providing quantitative, spatially resolved information on cells and their constituent molecules from the sub-micron to the whole-organ scale. Here we explore the application of spatial statistics to the cellular relationships within tissue microscopy data and discuss how spatial statistics offers cytometry a powerful yet underused mathematical tool set for which the required data are readily captured using standard protocols and microscopy equipment. We also highlight the often-overlooked need to carefully consider the structural heterogeneity of tissues in terms of the applicability of different statistical measures and their accuracy and demonstrate how spatial analyses offer a great deal more than just basic quantification of biological variance. Ultimately, we highlight how statistical modeling can help reveal the hierarchical spatial processes that connect the properties of individual cells to the establishment of biological function.

Comprehensive interpretation of in vitro micronucleus test results for 292 chemicals: from hazard identification to risk assessment application.

Risk assessments are increasingly reliant on information from in vitro assays. The in vitro micronucleus test (MNvit) is a genotoxicity test that detects chromosomal abnormalities, including chromosome breakage (clastogenicity) and/or whole chromosome loss (aneugenicity). In this study, MNvit datasets for 292 chemicals, generated by the US EPA's ToxCast program, were evaluated using a decision tree-based pipeline for hazard identification. Chemicals were tested with 19 concentrations (n = 1) up to 200 µM, in the presence and absence of Aroclor 1254-induced rat liver S9. To identify clastogenic chemicals, %MN values at each concentration were compared to a distribution of batch-specific solvent controls; this was followed by cytotoxicity assessment and benchmark concentration (BMC) analyses. The approach classified 157 substances as positives, 25 as negatives, and 110 as inconclusive. Using the approach described in Bryce et al. (Environ Mol Mutagen 52:280-286, 2011), we identified 15 (5%) aneugens. IVIVE (in vitro to in vivo extrapolation) was employed to convert BMCs into administered equivalent doses (AEDs). Where possible, AEDs were compared to points of departure (PODs) for traditional genotoxicity endpoints; AEDs were generally lower than PODs based on in vivo endpoints. To facilitate interpretation of in vitro MN assay concentration-response data for risk assessment, exposure estimates were utilized to calculate bioactivity exposure ratio (BER) values. BERs for 50 clastogens and two aneugens had AEDs that approached exposure estimates (i.e., BER < 100); these chemicals might be considered priorities for additional testing. This work provides a framework for the use of high-throughput in vitro genotoxicity testing for priority setting and chemical risk assessment.

Discovery and Characterization of an Aberrant Small Form of Glycoprotein I of Herpes Simplex Virus Type I in Cell Culture.

The 380-to-393-amino-acid glycoprotein I (gI) encoded by herpes simplex virus 1 (HSV-1) is a critical mediator for viral cell-to-cell spread and syncytium formation. Here we report a previously unrecognized aberrant form of gI in HSV-1-infected cells. Production of this molecule is independent of cell type and viral strains. It had an unexpected gel migration size of approximately 23 kDa, was packaged into viral particles, and could be coimmunoprecipitated by antibodies to both N and C termini of gI. Deep sequencing failed to detect alternative RNA splicing, and the invitro transcribed full-length mRNA gave rise to the 23 kDa protein in transfected cells. Combined mass spectrometry and antibody probing analyses detected peptide information across different regions of gI, suggesting the possibility of a full-length gI but with abnormal migration behavior. In line with this notion, the HA insertion mutagenesis revealed a stable fold in the gI extracellular region aa.38-196 resistant to denaturing conditions, whereas small deletions within this region failed the antibodies to detect the fast, but not the slow-moving species of gI. It is also intriguing that the structure could be perturbed to some extent by a gBsyn mutation, leading to exposure or shielding of the gI epitopes. Thus, the HSV-1 gI apparently adopts a very stable fold in its natural form, rendering it an unusual biophysical property. Our findings provide novel insight into the biological properties of HSV gI and have important implications in understanding the viral spread and pathogenesis. IMPORTANCE The HSV-1 gI is required for viral cell-to-cell spread within the host, but its behavior during infection has remained poorly defined. Along with the classic 66 kDa product, here we report a previously unrecognized, approximately 23 kDa form of gI. Biochemical and genetics analyses revealed that this molecule represents the full-length form of gI but adopts a stable fold in its extracellular domain that is resistant to denatured conditions, thus contributing to the aberrant migration rate. Our results revealed a novel property of HSV-1 gI and have important implications in understanding viral pathogenesis.

Developing ovine mammary terminal duct lobular units have a dynamic mucosal and stromal immune microenvironment.

The human breast and ovine mammary gland undergo striking levels of postnatal development, leading to formation of terminal duct lobular units (TDLUs). Here we interrogate aspects of sheep TDLU growth as a model of breast development and to increase understanding of ovine mammogenesis. The distributions of epithelial nuclear Ki67 positivity differ significantly between younger and older lambs. Ki67 expression is polarised to the leading edge of the developing TDLUs. Intraepithelial ductal macrophages exhibit periodicity and considerably increased density in lambs approaching puberty. Stromal macrophages are more abundant centrally than peripherally. Intraepithelial T lymphocytes are more numerous in older lambs. Stromal hotspots of Ki67 expression colocalize with immune cell aggregates that exhibit distinct organisation consistent with tertiary lymphoid structures. The lamb mammary gland thus exhibits a dynamic mucosal and stromal immune microenvironment and constitutes a valuable model system that provides new insights into postnatal breast development.

Formulation of Metal-Organic Framework-Based Drug Carriers by Controlled Coordination of Methoxy PEG Phosphate: Boosting Colloidal Stability and Redispersibility.

Metal-organic framework nanoparticles (nanoMOFs) have been widely studied in biomedical applications. Although substantial efforts have been devoted to the development of biocompatible approaches, the requirement of tedious synthetic steps, toxic reagents, and limitations on the shelf life of nanoparticles in solution are still significant barriers to their translation to clinical use. In this work, we propose a new postsynthetic modification of nanoMOFs with phosphate-functionalized methoxy polyethylene glycol (mPEG-PO3) groups which, when combined with lyophilization, leads to the formation of redispersible solid materials. This approach can serve as a facile and general formulation method for the storage of bare or drug-loaded nanoMOFs. The obtained PEGylated nanoMOFs show stable hydrodynamic diameters, improved colloidal stability, and delayed drug-release kinetics compared to their parent nanoMOFs. Ex situ characterization and computational studies reveal that PEGylation of PCN-222 proceeds in a two-step fashion. Most importantly, the lyophilized, PEGylated nanoMOFs can be completely redispersed in water, avoiding common aggregation issues that have limited the use of MOFs in the biomedical field to the wet form-a critical limitation for their translation to clinical use as these materials can now be stored as dried samples. The in vitro performance of the addition of mPEG-PO3 was confirmed by the improved intracellular stability and delayed drug-release capability, including lower cytotoxicity compared with that of the bare nanoMOFs. Furthermore, z-stack confocal microscopy images reveal the colocalization of bare and PEGylated nanoMOFs. This research highlights a facile PEGylation method with mPEG-PO3, providing new insights into the design of promising nanocarriers for drug delivery.

In Situ Pseudopotentials for Electronic Structure Theory.

We present a general method of constructing in situ pseodopotentials from first-principles, all-electron, and full-potential electronic structure calculations of a solid. The method is applied to bcc Na, at low-temperature equilibrium volume. The essential steps of the method involve (i) calculating an all-electron Kohn-Sham eigenstate, (ii) replacing the oscillating part of the wave function (inside the muffin-tin spheres) of this state, with a smooth function, (iii) representing the smooth wave function in a Fourier series, and (iv) inverting the Kohn-Sham equation, to extract the pseudopotential that produces the state generated in steps i-iii. It is shown that an in situ pseudopotential can reproduce an all-electron full-potential eigenvalue up to the sixth significant digit. A comparison of the all-electron theory, in situ pseudopotential theory, and the standard nonlocal pseudopotential theory demonstrates good agreement, e.g., in the energy dispersion of the 3s band state of bcc Na.

Inter-laboratory automation of the in vitro micronucleus assay using imaging flow cytometry and deep learning.

The in vitro micronucleus assay is a globally significant method for DNA damage quantification used for regulatory compound safety testing in addition to inter-individual monitoring of environmental, lifestyle and occupational factors. However, it relies on time-consuming and user-subjective manual scoring. Here we show that imaging flow cytometry and deep learning image classification represents a capable platform for automated, inter-laboratory operation. Images were captured for the cytokinesis-block micronucleus (CBMN) assay across three laboratories using methyl methanesulphonate (1.25-5.0 µg/mL) and/or carbendazim (0.8-1.6 µg/mL) exposures to TK6 cells. Human-scored image sets were assembled and used to train and test the classification abilities of the "DeepFlow" neural network in both intra- and inter-laboratory contexts. Harnessing image diversity across laboratories yielded a network able to score unseen data from an entirely new laboratory without any user configuration. Image classification accuracies of 98%, 95%, 82% and 85% were achieved for 'mononucleates', 'binucleates', 'mononucleates with MN' and 'binucleates with MN', respectively. Successful classifications of 'trinucleates' (90%) and 'tetranucleates' (88%) in addition to 'other or unscorable' phenotypes (96%) were also achieved. Attempts to classify extremely rare, tri- and tetranucleated cells with micronuclei into their own categories were less successful (≤ 57%). Benchmark dose analyses of human or automatically scored micronucleus frequency data yielded quantitation of the same equipotent concentration regardless of scoring method. We conclude that this automated approach offers significant potential to broaden the practical utility of the CBMN method across industry, research and clinical domains. We share our strategy using openly-accessible frameworks.

Empirical comparison of genotoxic potency estimations: the in vitro DNA-damage ToxTracker endpoints versus the in vivo micronucleus assay.

Genetic toxicology is an essential component of compound safety assessment. In the face of a barrage of new compounds, higher throughput, less ethically divisive in vitro approaches capable of effective, human-relevant hazard identification and prioritisation are increasingly important. One such approach is the ToxTracker assay, which utilises murine stem cell lines equipped with green fluorescent protein (GFP)-reporter gene constructs that each inform on distinct aspects of cellular perturbation. Encouragingly, ToxTracker has shown improved sensitivity and specificity for the detection of known in vivo genotoxicants when compared to existing 'standard battery' in vitro tests. At the current time however, quantitative genotoxic potency correlations between ToxTracker and well-recognised in vivo tests are not yet available. Here we use dose-response data from the three DNA-damage-focused ToxTracker endpoints and from the in vivo micronucleus assay to carry out quantitative, genotoxic potency estimations for a range of aromatic amine and alkylating agents using the benchmark dose (BMD) approach. This strategy, using both the exponential and the Hill BMD model families, was found to produce robust, visually intuitive and similarly ordered genotoxic potency rankings for 17 compounds across the BSCL2-GFP, RTKN-GFP and BTG2-GFP ToxTracker endpoints. Eleven compounds were similarly assessed using data from the in vivo micronucleus assay. Cross-systems genotoxic potency correlations for the eight matched compounds demonstrated in vitro-in vivo correlation, albeit with marked scatter across compounds. No evidence for distinct differences in the sensitivity of the three ToxTracker endpoints was found. The presented analyses show that quantitative potency determinations from in vitro data enable more than just qualitative screening and hazard identification in genetic toxicology.

Induction of Rod-Shaped Structures by Herpes Simplex Virus Glycoprotein I.

The envelope glycoprotein I (gI) of herpes simplex virus 1 (HSV-1) is a critical mediator of virus-induced cell-to-cell spread and cell-cell fusion. Here, we report a previously unrecognized property of this molecule. In transfected cells, the HSV-1 gI was discovered to induce rod-shaped structures that were uniform in width but variable in length. Moreover, the gI within these structures was conformationally different from the typical form of gI, as a previously used monoclonal antibody mAb3104 and a newly made peptide antibody to the gI extracellular domain (ECD) (amino acids [aa] 110 to 202) both failed to stain the long rod-shaped structures, suggesting the formation of a higher-order form. Consistent with this observation, we found that gI could self-interact and that the rod-shaped structures failed to recognize glycoprotein E, the well-known binding partner of gI. Further analyses by deletion mutagenesis and construction of chimeric mutants between gI and gD revealed that the gI ECD is the critical determinant, whereas the transmembrane domain served merely as an anchor. The critical amino acids were subsequently mapped to proline residues 184 and 188 within a conserved PXXXP motif. Reverse genetics analyses showed that the ability to induce a rod-shaped structure was not required for viral replication and spread in cell culture but rather correlated positively with the capability of the virus to induce cell fusion in the UL24syn background. Together, this work discovered a novel feature of HSV-1 gI that may have important implications in understanding gI function in viral spread and pathogenesis.IMPORTANCE The HSV-1 gI is required for viral cell-to-cell spread within the host, but the molecular mechanisms of how gI exactly works have remained poorly understood. Here, we report a novel property of this molecule, namely, induction of rod-shaped structures, which appeared to represent a higher-order form of gI. We further mapped the critical residues and showed that the ability of gI to induce rod-shaped structures correlated well with the capability of HSV-1 to induce cell fusion in the UL24syn background, suggesting that the two events may have an intrinsic link. Our results shed light on the biological properties of HSV-1 gI and may have important implications in understanding viral pathogenesis.

Gastrointestinal Absorption and Toxicity of Nanoparticles and Microparticles: Myth, Reality and Pitfalls explored through Titanium Dioxide.

Daily oral exposure to vast numbers (>1013/adult/day) of micron or nano-sized persistent particles has become the norm for many populations. Significant airborne particle exposure is deleterious, so what about ingestion? Titanium dioxide in food grade form (fgTiO2) , which is an additive to some foods, capsules, tablets and toothpaste, may provide clues. Certainly, exposed human populations accumulate these particles in specialised intestinal cells at the base of large lymphoid follicles (Peyer's patches) and it's likely that a degree of absorption goes beyond this- i.e. lymphatics to blood circulation to tissues. We critically review the evidence and pathways. Regarding potential adverse effects, our primary message, for today's state-of-art, is that in vivo models have not been good enough and at times woeful. We provide a 'caveats list' to improve approaches and experimentation and illustrate why studies on biomarkers of particle uptake, and lower gut/mesenteric lymph nodes as targets, should be prioritized.

A Murine Oral-Exposure Model for Nano- and Micro-Particulates: Demonstrating Human Relevance with Food-Grade Titanium Dioxide.

Human exposure to persistent, nonbiological nanoparticles and microparticles via the oral route is continuous and large scale (1012 -1013 particles per day per adult in Europe). Whether this matters or not is unknown but confirmed health risks with airborne particle exposure warns against complacency. Murine models of oral exposure will help to identify risk but, to date, lack validation or relevance to humans. This work addresses that gap. It reports i) on a murine diet, modified with differing concentrations of the common dietary particle, food grade titanium dioxide (fgTiO2 ), an additive of polydisperse form that contains micro- and nano-particles, ii) that these diets deliver particles to basal cells of intestinal lymphoid follicles, exactly as is reported as a "normal occurrence" in humans, iii) that confocal reflectance microscopy is the method of analytical choice to determine this, and iv) that food intake, weight gain, and Peyer's patch immune cell profiles, up to 18 weeks of feeding, do not differ between fgTiO2 -fed groups or controls. These findings afford a human-relevant and validated oral dosing protocol for fgTiO2 risk assessment as well as provide a generalized platform for application to oral exposure studies with nano- and micro-particles.