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Funder: Canada Research Chairs; doi: http://dx.doi.org/10.13039/501100001804, 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.

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.

Funder: University of Cambridge | Girton College, University of Cambridge (Girton); doi: https://doi.org/10.13039/501100000621, Funder: University of Cambridge Herchel-Smith Fund, Funder: British Veterinary Association Animal Welfare Foundation Norman Hayward Fund ref NHF_2016_03_KH, 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.

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. Supplementary Information The online version contains supplementary material available at 10.1007/s00204-021-03113-0.

Summary For many nanoparticle applications it is important to understand dispersion in liquids. For nanomedicinal and nanotoxicological research this is complicated by the often complex nature of the biological dispersant and ultimately this leads to severe limitations in the analysis of the nanoparticle dispersion by light scattering techniques. Here we present an alternative analysis and associated workflow which utilises electron microscopy. The need to collect large, statistically relevant datasets by imaging vacuum dried, plunge frozen aliquots of suspension was accomplished by developing an automated STEM imaging protocol implemented in an SEM fitted with a transmission detector. Automated analysis of images of agglomerates was achieved by machine learning using two free open‐source software tools: CellProfiler and ilastik. The specific results and overall workflow described enable accurate nanoparticle agglomerate analysis of particles suspended in aqueous media containing other potential confounding components such as salts, vitamins and proteins. Lay Description In order to further advance studies in both nanomedicine and nanotoxicology, we need to continue to understand the dispersion of nanoparticles in biological fluids. These biological environments often contain a number of components such as salts, vitamins and proteins which can lead to difficulties when using traditional techniques to monitor dispersion. Here we present an alternative analysis which utilises electron microscopy. In order to use this approach statistically relevant large image datasets were collected from appropriately prepared samples of nanoparticle suspensions by implementing an automated imaging protocol. Automated analysis of these images was achieved through machine learning using two readily accessible freeware; CellProfiler and ilastik. The workflow presented enables accurate nanoparticle dispersion analysis of particles suspended in more complex biological media.

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.

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.

Glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1) plays a key role in multiple events during infection including virus entry, cell-to-cell spread, and virus-induced syncytia formation. Here, we provide evidence that an arginine/lysine cluster located at the transmembrane-cytoplasm interface of gD critically contributes to viral spread and cell-cell fusion. Our studies began with the discovery that packaging of gD into virions is almost completely blocked in the absence of tegument protein UL16. We subsequently identified a novel, direct, and regulated interaction between UL16 and gD, but this was not important for syncytia formation. However, a mutational analysis of the membrane-proximal basic residues of gD revealed that they are needed for the gBsyn phenotype, salubrinal-induced fusion of HSV-infected cells, and cell-to-cell spread. Finally, we found that these same gD tail basic residues are not required for cell fusion induced by a gKsyn variant.

Thein vitromicronucleus 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 dose 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.

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.

Immunofluorescence microscopy is an essential tool for tissue-based research, yet data reporting is almost always qualitative. Quantification of images, at the per-cell level, enables "flow cytometry-type" analyses with intact locational data but achieving this is complex. Gastrointestinal tissue, for example, is highly diverse: from mixed-cell epithelial layers through to discrete lymphoid patches. Moreover, different species (e.g., rat, mouse, and humans) and tissue preparations (paraffin/frozen) are all commonly studied. Here, using field-relevant examples, we develop open, user-friendly methodology that can encompass these variables to provide quantitative tissue microscopy for the field. Antibody-independent cell labeling approaches, compatible across preparation types and species, were optimized. Per-cell data were extracted from routine confocal micrographs, with semantic machine learning employed to tackle densely packed lymphoid tissues. Data analysis was achieved by flow cytometry-type analyses alongside visualization and statistical definition of cell locations, interactions and established microenvironments. First, quantification of Escherichia coli passage into human small bowel tissue, following Ussing chamber incubations exemplified objective quantification of rare events in the context of lumen-tissue crosstalk. Second, in rat jejenum, precise histological context revealed distinct populations of intraepithelial lymphocytes between and directly below enterocytes enabling quantification in context of total epithelial cell numbers. Finally, mouse mononuclear phagocyte-T cell interactions, cell expression and significant spatial cell congregations were mapped to shed light on cell-cell communication in lymphoid Peyers patch. Accessible, quantitative tissue microscopy provides a new window-of-insight to diverse questions in gastroenterology. It can also help combat some of the data reproducibility crisis associated with antibody technologies and over-reliance on qualitative microscopy. (c) 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry. Funding Agencies|UK Medical Research CouncilMedical Research Council UK (MRC) [MR/R005699/1]; UK Engineering and Physical Sciences Research CouncilEngineering & Physical Sciences Research Council (EPSRC) [EP/H008683/1]; UK Biotechnology and Biological Sciences Research CouncilBiotechnology and Biological Sciences Research Council (BBSRC) [BB/P026818/1]; Girton College; University of Cambridge Herchel-Smith Fund

The human breast and ovine mammary gland undergo a striking degree of postnatal development, leading to formation of terminal duct lobular units (TDLUs). In this study we interrogated aspects of sheep TDLU growth to increase understanding of ovine mammogenesis and as a model for the study of breast development. Mammary epithelial proliferation is significantly higher in lambs less than two months old than in peri-pubertal animals. Ki67 expression is polarized to the leading edge of the developing TDLUs. Intraepithelial ductal macrophages exhibit striking periodicity and significantly increased density in lambs approaching puberty. Stromal macrophages are more abundant centrally than peripherally. The developing ovine mammary gland is infiltrated by intraepithelial and stromal T lymphocytes that are significantly more numerous in older lambs. In the stroma, hotspots of Ki67 expression colocalize with large aggregates of lymphocytes and macrophages. Multifocally these aggregates exhibit distinct organization consistent with tertiary lymphoid structures. The lamb mammary gland thus exhibits a dynamic mucosal and stromal immune microenvironment and, as such, constitutes a valuable model system that provides new insights into postnatal breast development.Summary statementDevelopment of terminal duct lobular units in the sheep mammary gland involves distinct growth phases and macrophage and lymphocyte fluxes. Tertiary lymphoid structures are present subjacent to the mucosal epithelium.

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.

Background The multifaceted interactions between gastrointestinal (GI) helminth parasites, host gut microbiota and immune system are emerging as a key area of research within the field of host-parasite relationships. In spite of the plethora of data available on the impact that GI helminths exert on the composition of the gut microflora, whether alterations of microbial profiles are caused by direct parasite-bacteria interactions or, indirectly, by alterations of the GI environment (e.g. mucosal immunity) remains to be determined. Furthermore, no data is thus far available on the downstream roles that qualitative and quantitative changes in gut microbial composition play in the overall pathophysiology of parasite infection and disease. Results In this study, we investigated the fluctuations in microbiota composition and local immune microenvironment of sheep vaccinated against, and experimentally infected with, the ‘brown stomach worm’ Teladorsagia circumcincta, a parasite of worldwide socio-economic significance. We compared the faecal microbial profiles of vaccinated and subsequently infected sheep with those obtained from groups of unvaccinated/infected and unvaccinated/uninfected animals. We show that alterations of gut microbial composition are associated mainly with parasite infection, and that this involves the expansion of populations of bacteria with known pro-inflammatory properties that may contribute to the immunopathology of helminth disease. Using novel quantitative approaches for the analysis of confocal microscopy-derived images, we also show that gastric tissue infiltration of T cells is driven by parasitic infection rather than anti-helminth vaccination. Conclusions Teladorsagia circumcincta infection leads to an expansion of potentially pro-inflammatory gut microbial species and abomasal T cells. This data paves the way for future experiments aimed to determine the contribution of the gut flora to the pathophysiology of parasitic disease, with the ultimate aim to design and develop novel treatment/control strategies focused on preventing and/or restricting bacterial-mediated inflammation upon infection by GI helminths.

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., Medical Research Council [grant number MR/R005699/1], Brazilian Federal Agency for Support and Evaluation of Graduate Education - CAPES [grant number 88881.127953/2016-01], Girton College and the University of Cambridge Herchel-Smith Fellowships

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.

Introduction Soluble copper that can be acquired by bacteria is toxic and therefore antimicrobial. Whether nanostructured copper materials, in either disperse or agglomerated form, have antimicrobial impact, aside from that of their dissolution products, is not clear and was herein addressed. Methods We took five nanostructured copper materials, two metallic, and three oxo-hydroxides with one of these being silicate-substituted. Four agglomerated in the bacterial growth media whilst the silicate-substituted material remained disperse and small (6.5 nm diameter). Antibacterial activity against E. coli was assessed with copper phase distribution measured over time. Using the dose of soluble copper, and benchmark dose non-linear regression modelling, we determined how well this phase predicted antimicrobial activity. Finally, we used Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) analysis to investigate whether membrane adhesion effects by copper were plausible or if intracellular uptake most likely explained the bacterial impact of copper. Results Comparison over time of antimicrobial activity against particulate or soluble phases of the aquated materials clearly demonstrated that soluble copper but not particulate forms were associated with inhibition of bacterial growth. Indeed, the benchmark dose modelling showed the soluble dose required to cause a 50% reduction in E. coli growth was strongly clustered – for all particle formulations – at 14.5 mg/L (10–19 mg/L 90% confidence interval). By comparison, total copper levels associated with the same reduction in viability varied widely (45–549 mg/L). Finally, in favour of this soluble product dominance in terms of antimicrobial activity, copper had low association with bacterial membrane (something both soluble and particulate materials could do) but showed high intra-bacterial levels (something only soluble copper could do). Conclusion Taken together our data show that it is the uptake of soluble but not particulate copper, and the intracellular loading not just contact and membrane association, that drives copper toxicity to bacteria. Therapeutic strategies for novel antimicrobial copper compounds should consider these findings.

In vitro gene expression signatures to predict toxicological responses can provide mechanistic context for regulatory testing. We previously developed the TGx-28.65 genomic biomarker from a database of gene expression profiles derived from human TK6 cells exposed to 28 well-known compounds. The biomarker comprises 65 genes that can classify chemicals as DNA damaging or non-DNA damaging. In this study, we applied the TGx-28.65 genomic biomarker in parallel with the in vitro micronucleus (MN) assay to determine if two chemicals of regulatory interest at Health Canada, disperse orange (DO: the orange azo dye 3-[[4-[(4-Nitrophenyl)azo]phenyl] benzylamino]propanenitrile) and 1,2,4-benzenetriol (BT: a metabolite of benzene) are genotoxic or non-genotoxic. Both chemicals caused dose-dependent declines in relative survival and increases in apoptosis. A strong significant increase in MN induction was observed for all concentrations of BT; the top two concentrations of DO also caused a statistically significant increase in MN, but these increases were

There is growing interest in quantitative analysis of in vivo genetic toxicity dose-response data, and use of point-of-departure (PoD) metrics such as the benchmark dose (BMD) for human health risk assessment (HHRA). Currently, multiple transgenic rodent (TGR) assay variants, employing different rodent strains and reporter transgenes, are used for the assessment of chemically-induced genotoxic effects in vivo. However, regulatory issues arise when different PoD values (e.g., lower BMD confidence intervals or BMDLs) are obtained for the same compound across different TGR assay variants. This study therefore employed the BMD approach to examine the ability of different TGR variants to yield comparable genotoxic potency estimates. Review of over 2000 dose-response datasets identified suitably-matched dose-response data for three compounds (ethyl methanesulfonate or EMS, N-ethyl-N-nitrosourea or ENU, and dimethylnitrosamine or DMN) across four commonly-used murine TGR variants (Muta™Mouse lacZ, Muta™Mouse cII, gpt delta and BigBlue® lacI). Dose-response analyses provided no conclusive evidence that TGR variant choice significantly influences the derived genotoxic potency estimate. This conclusion was reliant upon taking into account the importance of comparing BMD confidence intervals as opposed to directly comparing PoD values (e.g., comparing BMDLs). Comparisons with earlier works suggested that with respect to potency determination, tissue choice is potentially more important than choice of TGR assay variant. Scoring multiple tissues selected on the basis of supporting toxicokinetic information is therefore recommended. Finally, we used typical within-group variances to estimate preliminary endpoint-specific benchmark response (BMR) values across several TGR variants/tissues. We discuss why such values are required for routine use of genetic toxicity PoDs for HHRA. Environ. Mol. Mutagen. 58:632-643, 2017. © 2017 Her Majesty the Queen in Right of Canada. Environmental and Molecular Mutagenesis Published by Wiley Periodicals, Inc.

Silicon (Si) has long been known to play a major physiological and structural role in certain organisms, including diatoms, sponges, and many higher plants, leading to the recent identification of multiple proteins responsible for Si transport in a range of algal and plant species. In mammals, despite several convincing studies suggesting that silicon is an important factor in bone development and connective tissue health, there is a critical lack of understanding about the biochemical pathways that enable Si homeostasis. Here we report the identification of a mammalian efflux Si transporter, namely Slc34a2 (also termed NaPiIIb), a known sodium-phosphate cotransporter, which was upregulated in rat kidney following chronic dietary Si deprivation. Normal rat renal epithelium demonstrated punctate expression of Slc34a2, and when the protein was heterologously expressed in Xenopus laevis oocytes, Si efflux activity (i.e., movement of Si out of cells) was induced and was quantitatively similar to that induced by the known plant Si transporter OsLsi2 in the same expression system. Interestingly, Si efflux appeared saturable over time, but it did not vary as a function of extracellular [Formula: see text] or Na+ concentration, suggesting that Slc34a2 harbors a functionally independent transport site for Si operating in the reverse direction to the site for phosphate. Indeed, in rats with dietary Si depletion-induced upregulation of transporter expression, there was increased urinary phosphate excretion. This is the first evidence of an active Si transport protein in mammals and points towards an important role for Si in vertebrates and explains interactions between dietary phosphate and silicon.

For phenotypic behavior to be understood in the context of cell lineage and local environment, properties of individual cells must be measured relative to population-wide traits. However, the inability to accurately identify, track and measure thousands of single cells via high-throughput microscopy has impeded dynamic studies of cell populations. We demonstrate unique labeling of cells, driven by the heterogeneous random uptake of fluorescent nanoparticles of different emission colors. By sequentially exposing a cell population to different particles, we generated a large number of unique digital codes, which corresponded to the cell-specific number of nanoparticle-loaded vesicles and were visible within a given fluorescence channel. When three colors are used, the assay can self-generate over 17,000 individual codes identifiable using a typical fluorescence microscope. The color-codes provided immediate visualization of cell identity and allowed us to track human cells with a success rate of 78% across image frames separated by 8 h.

Understanding nanoparticle uptake by biological cells is fundamentally important to wide-ranging fields from nanotoxicology to drug delivery. It is now accepted that the arrival of nanoparticles at the cell is an extremely complicated process, shaped by many factors including unique nanoparticle physico-chemical characteristics, protein-particle interactions and subsequent agglomeration, diffusion and sedimentation. Sequentially, the nanoparticle internalisation process itself is also complex, and controlled by multiple aspects of a cell’s state. Despite this multitude of factors, here we demonstrate that the statistical distribution of the nanoparticle dose per endosome is independent of the initial administered dose and exposure duration. Rather, it is the number of nanoparticle containing endosomes that are dependent on these initial dosing conditions. These observations explain the heterogeneity of nanoparticle delivery at the cellular level and allow the derivation of simple, yet powerful probabilistic distributions that accurately predict the nanoparticle dose delivered to individual cells across a population., Cellular uptake of nanoparticles is highly variable between individual cells in a population. Here, the authors show that this heterogeneity is a result of varying numbers of nanoparticle-containing endosomes while the nanoparticle dose per endosome remains constant.

Like all the herpesviruses, herpes simplex virus encodes machinery that enables it to move through cell junctions to avoid neutralizing antibodies. This cell-to-cell spread mechanism requires the viral fusion machinery (gD, gH/gL, and gB) and numerous accessory proteins. Of all of these, minor alterations to only four proteins (gB, gK, UL20, or UL24) will dysregulate the fusion machinery, allowing the formation of syncytia. In contrast, removal of individual accessory proteins will block cell-to-cell spread, forcing the virus to transmit in a cell-free manner. In the context of a Syn variant, removal of a required accessory protein will block cell fusion, again forcing cell-free spread. This has been investigated most thoroughly for gBsyn variants, which lose their syncytial phenotype in the absence of several accessory proteins, including gE, gI, UL16, and UL21, which are known to physically interact. Recently it was found that UL21 is not needed for gKsyn-, UL20syn-, or UL24syn-induced cell fusion, and hence it was of interest to ascertain whether gE, gI, and UL16 are required for Syn variants other than gBsyn. Null mutants of these were each combined with seven syncytial variants distributed among gK, UL20, and UL24. Surprisingly, very different patterns of accessory protein requirements were revealed. Indeed, for the three gKsyn variants tested, two different patterns were found. Also, three mutants were able to replicate without causing cytopathic effects. These findings show that mutations that produce Syn variants dysregulate the cell-to-cell-spread machinery in unique ways and provide clues for elucidating how this virus moves between cells. IMPORTANCE Approximately 2/3 of adults worldwide are latently infected with herpes simplex virus 1. Upon reactivation, the virus has the ability to evade neutralizing antibodies by moving through cell junctions, but the mechanism of direct cell-to-cell spread is poorly understood. The machinery that assembles between cells includes the viral fusion proteins and various accessory proteins that prevent cells from fusing. Alterations in four proteins will dysregulate the machinery, allowing neighboring cells to fuse to make syncytia, but this can be prevented by removing various individual accessory proteins to further disable the machinery. Previously, the accessory protein UL21 was found to be important for the activity of some syncytial variants but not others. In this study, we discovered that UL16, gE, and gI all act differently in how they control the fusion machinery. A better understanding of the mechanism of cell-to-cell spread may enable the development of drugs that block it.

All herpesviruses have mechanisms for passing through cell junctions, which exclude neutralizing antibodies and offer a clear path to neighboring, uninfected cells. In the case of herpes simplex virus type 1 (HSV-1), direct cell-to-cell transmission takes place between epithelial cells and sensory neurons, where latency is established. The spreading mechanism is poorly understood, but mutations in four different HSV-1 genes can dysregulate it, causing neighboring cells to fuse to produce syncytia. Because the host proteins involved are largely unknown (other than the virus entry receptor), we were intrigued by an earlier discovery that cells infected with wild-type HSV-1 will form syncytia when treated with salubrinal. A biotinylated derivative of this drug was used to pull down cellular complexes, which were analyzed by mass spectrometry. One candidate was a protein tyrosine phosphatase (PTP1B), and although it ultimately proved not to be the target of salubrinal, it was found to be critical for the mechanism of cell-to-cell spread. In particular, a highly specific inhibitor of PTP1B (CAS 765317-72-4) blocked salubrinal-induced fusion, and by itself resulted in a dramatic reduction in the ability of HSV-1 to spread in the presence of neutralizing antibodies. The importance of this phosphatase was confirmed in the absence of drugs by using PTP1B-/- cells. Importantly, replication assays showed that virus titers were unaffected when PTP1B was inhibited or absent. Only cell-to-cell spread was altered. We also examined the effects of salubrinal and the PTP1B inhibitor on the four Syn mutants of HSV-1, and strikingly different responses were found. That is, both drugs individually enhanced fusion for some mutants and reduced fusion for others. PTP1B is the first host factor identified to be specifically required for cell-to-cell spread, and it may be a therapeutic target for preventing HSV-1 reactivation disease., Author summary It is estimated that 67% of the global population is infected with herpes simplex virus type 1 (HSV-1). This virus resides in sensory neurons in a quiescent state but periodically reactivates, producing virus particles that travel down the axon to infect epithelial cells of the skin, where it can be transmitted to additional people. To avoid neutralizing antibodies, herpesviruses have evolved mechanisms for moving directly from one cell to another through their sites of intimate contact; however, the mechanism of cell-to-cell spread is poorly understood. Studies of HSV-1 mutants have implicated numerous viral proteins, but the necessary cellular factors are unknown except for the one that the virus uses to enter cells. Our experiments have identified a cellular enzyme (PTP1B, a tyrosine phosphatase) that is dispensable for the production of infectious virions but is critically important for the cell-to-cell spreading mechanism. Promising drugs targeting PTP1B have already been tested in early clinical trials for possible treatment of obesity and type-2 diabetes, and thus, our study may have immediate utility for attenuating HSV-1 reactivation disease in immunocompromised patients.

Use of imaging flow cytometry to assess induced DNA damage via the cytokinesis block micronucleus (CBMN) assay has thus far been limited to radiation dosimetry in human lymphocytes using high end, 'ImageStream X' series imaging cytometers. Its potential to enumerate chemically induced DNA damage using in vitro cell lines remains unexplored. In the present manuscript, we investigate the more affordable FlowSight® imaging cytometry platform to assess in vitro micronucleus (MN) induction in the human lymphoblastoid TK6 and metabolically competent MCL-5 cells treated with Methyl Methane Sulfonate (MMS) (0-5 µg/ml), Carbendazim (0-1.6 µg/ml), and Benzo[a]Pyrene (B[a]P) (0-6.3 µg/ml) for a period of 1.5-2 cell-cycles. Cells were fixed, and nuclei and MN were stained using the fluorescent nuclear dye DRAQ5™. Image acquisition was carried out using a 20X objective on a FlowSight® imaging cytometer (Amnis, part of Merck Millipore) equipped with a 488 nm laser. Populations of ∼20000 brightfield cell images, alongside DRAQ5™ stained nuclei/MN were rapidly collected (≤10 min). Single, in-focus cells suitable for scoring were then isolated using the IDEAS® software. An overlay of the brightfield cell outlines and the DRAQ5 nuclear fluorescence was used to facilitate scoring of mono-, bi-, tri-, and tetra-nucleated cells with or without MN events and in context of the cytoplasmic boundary of the parent cell.To establish the potential of the FlowSight® platform, and to establish 'ground truth' cell classification for the supervised machine learning based scoring algorithm that represents the next stage of our project, the captured images were scored manually. Alongside, MN frequencies were also derived using the 'gold standard' light microscopy and manual scoring. A minimum of 3000 bi-nucleated cells were assessed using both approaches. Using the benchmark dose approach, the comparability of genotoxic potency estimations for the different compounds and cell lines was assessed across the two scoring platforms as highly similar. This study therefore provides essential proof-of-concept that FlowSight® imaging cytometry is capable of reproducing the results of 'gold standard' manual scoring by light microscopy. We conclude that, with the right automated scoring algorithm, imaging flow cytometry could revolutionise the reportability and scoring throughput of the CBMN assay.

Assessment of genetic toxicity and/or carcinogenic activity is an essential element of chemical screening programs employed to protect human health. Dose-response and gene mutation data are frequently analysed by industry, academia and governmental agencies for regulatory evaluations and decision making. Over the years, a number of efforts at different institutions have led to the creation and curation of databases to house genetic toxicology data, largely, with the aim of providing public access to facilitate research and regulatory assessments. This article provides a brief introduction to a new genetic toxicology portal called Mutation Analysis Informatics Tools (MutAIT) (www.mutait.org) that provides easy access to two of the largest genetic toxicology databases, the Mammalian Gene Mutation Database (MGMD) and TransgenicDB. TransgenicDB is a comprehensive collection of transgenic rodent mutation data initially compiled and collated by Health Canada. The updated MGMD contains approximately 50 000 individual mutation spectral records from the published literature. The portal not only gives access to an enormous quantity of genetic toxicology data, but also provides statistical tools for dose-response analysis and calculation of benchmark dose. Two important R packages for dose-response analysis are provided as web-distributed applications with user-friendly graphical interfaces. The 'drsmooth' package performs dose-response shape analysis and determines various points of departure (PoD) metrics and the 'PROAST' package provides algorithms for dose-response modelling. The MutAIT statistical tools, which are currently being enhanced, provide users with an efficient and comprehensive platform to conduct quantitative dose-response analyses and determine PoD values that can then be used to calculate human exposure limits or margins of exposure.

The application of flow cytometry as a scoring platform for both in vivo and in vitro micronucleus (MN) studies has enabled the efficient generation of high quality datasets suitable for comprehensive assessment of dose–response. Using this information, it is possible to obtain precise estimates of the clastogenic potency of chemicals. We illustrate this by estimating the in vivo and the in vitro potencies of seven model clastogenic agents (melphalan, chlorambucil, thiotepa, 1,3-propane sultone, hydroxyurea, azathioprine and methyl methanesulfonate) by deriving BMDs using freely available BMD software (PROAST). After exposing male rats for 3 days with up to nine dose levels of each individual chemical, peripheral blood samples were collected on Day 4. These chemicals were also evaluated for in vitro MN induction by treating TK6 cells with up to 20 concentrations in quadruplicate. In vitro MN frequencies were determined via flow cytometry using a 96-well plate autosampler. The estimated in vitro and in vivo BMDs were found to correlate to each other. The correlation showed considerable scatter, as may be expected given the complexity of the whole animal model versus the simplicity of the cell culture system. Even so, the existence of the correlation suggests that information on the clastogenic potency of a compound can be derived from either whole animal studies or cell culture-based models of chromosomal damage. We also show that the choice of the benchmark response, i.e. the effect size associated with the BMD, is not essential in establishing the correlation between both systems. Our results support the concept that datasets derived from comprehensive genotoxicity studies can provide quantitative dose–response metrics. Such investigational studies, when supported by additional data, might then contribute directly to product safety investigations, regulatory decision-making and human risk assessment.

Genetic toxicology testing has a crucial role in the safety assessment of new and existing substances of societal value by reducing/eliminating human exposure to potential somatic and germ cell mutagens. Genetic toxicology assays have historically been used in a qualitative manner to arrive at the binary decision of ‘yes’ or ‘no’ with regards to the mutagenic potential. However, the field is currently at a crossroads, with new methods being developed and new proposals being made to use genetic toxicity data in a more quantitative manner. Technological advances have made it possible to perform high-content, high-throughput and high-precision analysis to increase the number of “scored” events leading to increased statistical precision of the endpoint under evaluation. Automated flow cytometry and image analysis are providing significant advantages for the evaluation of gene mutations as well as cytogenetic damage both in vitro and in vivo. In addition, statistical methods such as the benchmark dose (BMD) approach can be used to identify point of departure (PoD) metrics for use in human health risk assessments, including estimation of reference dose (RfD) and margins of exposure (MOE) from in vivo data. Here we provide new data to compare different in vitro micronucleus approaches, observing that the flow based assay performs very well in defining a PoD for methyl methanesulfonate. We also present reanalysis of published in vivo Pig-a gene mutation data, to show how covariate analysis increases precision and reduces the effects of outliers when defining BMD values. Furthermore, we show how in vivo BMD metrics can be used to define RfD values, and then provide comparisons to other human exposure limit values such as permitted daily exposure (PDE). Finally, the principles of empirical correlation using BMD metrics are presented, with methods for derivation of BMD values for endpoint B, when using data from only endpoint A. These developments are opening the possibility of genetic toxicity data being used as an apical endpoint to define negligible risk in human health risk assessments. Expert groups consisting of stakeholders representing academia, industry and the government are now developing guidance on transforming genetic toxicology testing from a qualitative to a quantitative science, keeping in mind the 3R principles of animal welfare.

The initial goal of this study was to reexamine the requirement of UL21 for herpes simplex virus 1 (HSV-1) replication. Previous studies suggested that UL21 is dispensable for replication in cell cultures, but a recent report on HSV-2 challenges those findings. As was done for the HSV-2 study, a UL21-null virus was made and propagated on complementing cells to discourage selection of compensating mutations. This HSV-1 mutant was able to replicate in noncomplementing cells, even at a low multiplicity of infection (MOI), though a reduction in titer was observed. Also, increased proportions of empty capsids were observed in the cytoplasm, suggesting a role for UL21 in preventing their exit from the nucleus. Surprisingly, passage of the null mutant resulted in rapid outgrowth of syncytial (Syn) variants. This was unexpected because UL21 has been shown to be required for the Syn phenotype. However, earlier experiments made use of only the A855V syncytial mutant of glycoprotein B (gB), and the Syn phenotype can also be produced by substitutions in glycoprotein K (gK), UL20, and UL24. Sequencing of the syncytial variants revealed mutations in the gK locus, but UL21 was shown to be dispensable for UL20 Syn and UL24 Syn . To test whether UL21 is needed only for the A855V mutant, additional gB Syn derivatives were examined in the context of the null virus, and all produced lytic rather than syncytial sites of infection. Thus, UL21 is required only for the gB Syn phenotype. This is the first example of a differential requirement for a viral protein across the four syn loci. IMPORTANCE UL21 is conserved among alphaherpesviruses, but its role is poorly understood. This study shows that HSV-1 can replicate without UL21, although the virus titers are greatly reduced. The null virus had greater proportions of empty (DNA-less) capsids in the cytoplasm of infected cells, suggesting that UL21 may play a role in retaining them in the nucleus. This is consistent with reports showing UL21 to be capsid associated and localized to the nuclei of infected cells. UL21 also appears to be needed for viral membrane activities. It was found to be required for virus-mediated cell fusion, but only for mutants that harbor syncytial mutations in gB (not variants of gK, UL20, or UL24). The machinery needed for syncytial formation is similar to that needed for direct spread of the virus through cell junctions, and these studies show that UL21 is required for cell-to-cell spread even in the absence of syncytial mutations.

Understanding the delivered cellular dose of nanoparticles is imperative in nanomedicine and nanosafety, yet is known to be extremely complex because of multiple interactions between nanoparticles, their environment, and the cells. Here, we use 3-D reconstruction of agglomerates preserved by cryogenic snapshot sampling and imaged by electron microscopy to quantify the "bioavailable dose" that is presented at the cell surface and formed by the process of individual nanoparticle sequestration into agglomerates in the exposure media. Critically, using 20 and 40 nm carboxylated polystyrene-latex and 16 and 85 nm silicon dioxide nanoparticles, we show that abrupt, dose-dependent "tipping points" in agglomeration state can arise, subsequently affecting cellular delivery and increasing toxicity. These changes are triggered by shifts in the ratio of the total nanoparticle surface area to biomolecule abundance, with the switch to a highly agglomerated state effectively changing the test article midassay, challenging the dose-response paradigm for nanosafety experiments. By characterizing nanoparticle numbers per agglomerate, we show these tipping points can lead to the formation of extreme agglomeration states whereby 90% of an administered dose is contained and delivered to the cells by just the top 2% of the largest agglomerates. We thus demonstrate precise definition, description, and comparison of the nanoparticle dose formed in different experimental environments and show that this description is critical to understanding cellular delivery and toxicity. We further empirically "stress-test" the commonly used dynamic light scattering approach, establishing its limitations to present an analysis strategy that significantly improves the usefulness of this popular nanoparticle characterization technique.

The UL16 tegument protein of herpes simplex virus 1 (HSV-1) is conserved among all herpesviruses and plays many roles during replication. This protein has an N-terminal domain (NTD) that has been shown to bind to several viral proteins, including UL11, VP22, and glycoprotein E, and these interactions are negatively regulated by a C-terminal domain (CTD). Thus, in pairwise transfections, UL16 binding is enabled only when the CTD is absent or altered. Based on these results, we hypothesized that direct interactions occur between the NTD and the CTD. Here we report that the separated and coexpressed functional domains of UL16 are mutually responsive to each other in transfected cells and form complexes that are stable enough to be captured in coimmunoprecipitation assays. Moreover, we found that the CTD can associate with itself. To our surprise, the CTD was also found to contain a novel and intrinsic ability to localize to specific spots on mitochondria in transfected cells. Subsequent analyses of HSV-infected cells by immunogold electron microscopy and live-cell confocal imaging revealed a population of UL16 that does not merely accumulate on mitochondria but in fact makes dynamic contacts with these organelles in a time-dependent manner. These findings suggest that the domain interactions of UL16 serve to regulate not just the interaction of this tegument protein with its viral binding partners but also its interactions with mitochondria. The purpose of this novel interaction remains to be determined. IMPORTANCE The HSV-1-encoded tegument protein UL16 is involved in multiple events of the virus replication cycle, ranging from virus assembly to cell-cell spread of the virus, and hence it can serve as an important drug target. Unfortunately, a lack of both structural and functional information limits our understanding of this protein. The discovery of domain interactions within UL16 and the novel ability of UL16 to interact with mitochondria in HSV-infected cells lays a foundational framework for future investigations aimed at deciphering the structure and function of not just UL16 of HSV-1 but also its homologs in other herpesviruses.

The intrinsic limitations and pitfalls exhibited by two-dimensional (2D) in vitro studies in conjunction with the ethical issues surrounding in vivo (nano)toxicology testing on animals have prompted the need to develop three-dimensional (3D) models. This chapter discusses the problems faced when using monolayer cell cultures for safety assessment, which led to the rationale behind the development of 3D models for safety evaluation purposes. The additional benefits of 3D models for nanosafety are presented, coupled with an overview of some of the 3D in vitro models that have been developed to mimic the physiological environment of certain tissues/organs including liver, lung, colon, intestine and gastrointestinal tract.

UL16 is a tegument protein of herpes simplex virus (HSV) that is conserved among all members of the Herpesviridae , but its function is poorly understood. Previous studies revealed that UL16 is associated with capsids in the cytoplasm and interacts with the membrane protein UL11, which suggested a “bridging” function during cytoplasmic envelopment, but this conjecture has not been tested. To gain further insight, cells infected with UL16-null mutants were examined by electron microscopy. No defects in the transport of capsids to cytoplasmic membranes were observed, but the wrapping of capsids with membranes was delayed. Moreover, clusters of cytoplasmic capsids were often observed, but only near membranes, where they were wrapped to produce multiple capsids within a single envelope. Normal virion production was restored when UL16 was expressed either by complementing cells or from a novel position in the HSV genome. When the composition of the UL16-null viruses was analyzed, a reduction in the packaging of glycoprotein E (gE) was observed, which was not surprising, since it has been reported that UL16 interacts with this glycoprotein. However, levels of the tegument protein VP22 were also dramatically reduced in virions, even though this gE-binding protein has been shown not to depend on its membrane partner for packaging. Cotransfection experiments revealed that UL16 and VP22 can interact in the absence of other viral proteins. These results extend the UL16 interaction network beyond its previously identified binding partners to include VP22 and provide evidence that UL16 plays an important function at the membrane during virion production.

Genotoxicity tests have traditionally been used only for hazard identification, with qualitative dichotomous groupings being used to identify compounds that have the capacity to induce mutations and/or cytogenetic alterations. However, there is an increasing interest in employing quantitative analysis of in vivo dose-response data to derive point of departure (PoD) metrics that can be used to establish human exposure limits or margins of exposure (MOEs), thereby supporting human health risk assessments and regulatory decisions. This work is an extension of our companion article on in vitro dose-response analyses and outlines how the combined benchmark dose (BMD) approach across included covariates can be used to improve the analyses and interpretation of in vivo genetic toxicity dose-response data. Using the BMD-covariate approach, we show that empirical comparisons of micronucleus frequency dose-response data across multiple studies justifies dataset merging, with subsequent analyses improving the precision of BMD estimates and permitting attendant potency ranking of seven clastogens. Similarly, empirical comparisons of Pig-a mutant phenotype frequency data collected in males and females justified dataset merging across sex. This permitted more effective scrutiny regarding the effect of post-exposure sampling time on the mutagenicity of N-ethyl-N-nitrosourea observed in reticulocytes and erythrocytes in the Pig-a assay. The BMD-covariate approach revealed tissue-specific differences in the induction of lacZ transgene mutations in Muta™Mouse specimens exposed to benzo[a]pyrene (BaP), with the results permitting the formulation of mechanistic hypotheses regarding the observed potency ranking. Lastly, we illustrate how historical dose-response data for assessments that examined numerous doses (i.e. induced lacZ mutant frequency (MF) across 10 doses of BaP) can be used to improve the precision of BMDs derived from datasets with far fewer doses (i.e. lacZ MF for 3 doses of dibenz[a,h]anthracene). Collectively, the presented examples illustrate how innovative use of the BMD approach can permit refinement of the use of in vivo data; improving the efficacy of experimental animal use in genetic toxicology without sacrificing PoD precision.

Genetic toxicity testing has traditionally been used for hazard identification, with dichotomous classification of test results serving to identify genotoxic agents. However, the utility of genotoxicity data can be augmented by employing dose-response analysis and point of departure determination. Via interpolation from a fitted dose-response model, the benchmark dose (BMD) approach estimates the dose that elicits a specified (small) effect size. BMD metrics and their confidence intervals can be used for compound potency ranking within an endpoint, as well as potency comparisons across other factors such as cell line or exposure duration. A recently developed computational method, the BMD covariate approach, permits combined analysis of multiple dose-response data sets that are differentiated by covariates such as compound, cell type or exposure regime. The approach provides increased BMD precision for effective potency rankings across compounds and other covariates that pertain to a hypothesised mode of action (MOA). To illustrate these applications, the covariate approach was applied to the analysis of published in vitro micronucleus frequency dose-response data for ionising radiations, a set of aneugens, two mutagenic azo compounds and a topoisomerase II inhibitor. The ionising radiation results show that the precision of BMD estimates can be improved by employing the covariate method. The aneugen analysis provided potency groupings based on the BMD confidence intervals, and analyses of azo compound data from cells lines with differing metabolic capacity confirmed the influence of endogenous metabolism on genotoxic potency. This work, which is the first of a two-part series, shows that BMD-derived potency rankings can be employed to support MOA evaluations as well as facilitate read across to expedite chemical evaluations and regulatory decision-making. The follow-up (Part II) employs the combined covariate approach to analyse in vivo genetic toxicity dose-response data focussing on how improvements in BMD precision can impact the reduction and refinement of animal use in toxicological research.

Many applications in biomedical research require the long-term identification and tracking of cells over time. In previous work we have demonstrated that by sequentially dosing a cell population with different emission wavelength nanoparticles it is possible to use the random number of nanoparticle loaded vesicles generated by the cells as a barcode for individual cells within the population. In this paper we develop a simple model to describe the number of codes that can be generated using this sequential loading protocol. The methodology is validated by comparison with experiment and subsequently used to predict the effect of varying the number of colors used to encode the cells and also to assess the effect of misreading the cellular code due to errors in imaging the vesicles.

Background The rapid production and incorporation of engineered nanomaterials into consumer products alongside research suggesting nanomaterials can cause cell death and DNA damage (genotoxicity) makes in vitro assays desirable for nanosafety screening. However, conflicting outcomes are often observed when in vitro and in vivo study results are compared, suggesting more physiologically representative in vitro models are required to minimise reliance on animal testing. Method BASF Levasil® silica nanoparticles (16 and 85 nm) were used to adapt the 3D reconstructed skin micronucleus (RSMN) assay for nanomaterials administered topically or into the growth medium. 3D dose-responses were compared to a 2D micronucleus assay using monocultured human B cells (TK6) after standardising dose between 2D / 3D assays by total nanoparticle mass to cell number. Cryogenic vitrification, scanning electron microscopy and dynamic light scattering techniques were applied to characterise in-medium and air-liquid interface exposures. Advanced transmission electron microscopy imaging modes (high angle annular dark field) and X-ray spectrometry were used to define nanoparticle penetration / cellular uptake in the intact 3D models and 2D monocultured cells. Results For all 2D exposures, significant (p

Glycoprotein E (gE) of HSV plays a key role in cell-to-cell spread and virus-induced cell fusion. Here, we report that this function of gE requires the cooperation of tegument proteins UL11, UL16, and UL21. We found that the four proteins come together with very high efficiency to form a complex in transfected cells and in a manner that is regulated and coordinated. In particular, the inefficient interaction of UL16 with each membrane protein (UL11 and gE) observed in pairwise transfections became efficient when other binding partners were present. The significance of these interactions was revealed in studies of viral mutants, which showed that each of these tegument proteins is critical for processing, transport, and biological activity of gE. These findings provide insights into the mechanisms of how gE executes its function and also have implications in understanding HSV assembly and budding.

It is well known that proteins in the tegument (located between the viral capsid and envelope proteins) play critical roles in the assembly and budding of herpesviruses. Tegument proteins UL16 and UL11 of herpes simplex virus (HSV) are conserved among all the Herpesviridae . Although these proteins directly interact in vitro , UL16 was found to colocalize poorly with UL11 in cotransfected cells. To explain this discrepancy, we hypothesized that UL16 is initially made in an inactive form and is artificially transformed to the binding-competent state when cells are disrupted. Consistent with a regulated interaction, UL16 was able to fully colocalize with UL11 when a large C-terminal segment of UL16 was removed, creating mutant UL16(1-155). Moreover, membrane flotation assays revealed a massive movement of this mutant to the top of sucrose gradients in the presence of UL11, whereas both the full-length UL16 and the C-terminal fragment (residues 156 to 373) remained at the bottom. Further evidence for the presence of a C-terminal regulatory domain was provided by single-amino-acid substitutions at conserved cysteines (C269S, C271S, and C357S), which enabled the efficient interaction of full-length UL16 with UL11. Lastly, the binding site for UL11 was further mapped to residues 81 to 155, and to our surprise, the 5 Cys residues within UL16(1-155) are not required, even though the modification of free cysteines in UL16 with N -ethylmaleimide does in fact prevent binding. Collectively, these results reveal a regulatory function within the C-terminal region of UL16 that controls an N-terminal UL11-binding activity.

The UL11 tegument protein of herpes simplex virus plays a critical role in the secondary envelopment; however, the mechanistic details remain elusive. Here, we report a new function of UL11 in the budding process in which it directs efficient acquisition of glycoprotein E (gE) via a direct interaction. In vitro binding assays showed that the interaction required only the first 28, membrane-proximal residues of the cytoplasmic tail of gE, and the C-terminal 26 residues of UL11. A second, weaker binding site was also found in the N-terminal half of UL11. The significance of the gE-UL11 interaction was subsequently investigated with viral deletion mutants. In the absence of the gE tail, virion packaging of UL11, but not other tegument proteins such as VP22 and VP16, was reduced by at least 80%. Reciprocally, wild-type gE packaging was also drastically reduced by about 87% in the absence of UL11, and this defect could be rescued in trans by expressing U L 11 at the U L 35 locus. Surprisingly, a mutant that lacks the C-terminal gE-binding site of UL11 packaged nearly normal amounts of gE despite its strong interaction with the gE tail in vitro , indicating that the interaction with the UL11 N terminus may be important. Mutagenesis studies of the UL11 N terminus revealed that the association of UL11 with membrane was not required for this function. In contrast, the UL11 acidic cluster motif was found to be critical for gE packaging and was not replaceable with foreign acidic clusters. Together, these results highlight an important role of UL11 in the acquisition of glycoprotein-enriched lipid bilayers, and the findings may also have important implications for the role of UL11 in gE-mediated cell-to-cell spread.

Herpes simplex virus (HSV) contains more than 40 different virally encoded proteins that are found in three distinct layers: the capsid containing the viral DNA, the host-derived lipid envelope with embedded glycoproteins, and the tegument, an assortment of proteins located between the nucleocapsid and the envelope (22). While these regions are often discussed as separate structures, there is now clear evidence that the virion as a whole is a machine with interconnected parts that quickly rearrange on the inside in response to glycoprotein-binding events on the outside. Specifically, tegument protein UL16 is triggered to be released from the capsid when HSV attaches to host cells prior to membrane fusion, and the signal responsible for this can be sent in a cell-free manner by binding virions to immobilized heparin (21). It appears that glycoprotein C is involved in transmitting the signal (at least in a cell-free system), but all the other molecular “cogs” that drive this part of the HSV machine are unknown. To identify these components, we have been investigating UL16 and the network of molecular interactions in which it participates. Our interest in UL16 began when we identified it as a binding partner of UL11 (17), a small tegument protein (only 96 amino acids) that is conserved among all herpesviruses. UL11 is peripherally bound to membranes via two fatty acids, myristate and palmitate (16), and trafficks through lipid raft domains (6, 12). It accumulates at the trans-Golgi network (TGN), where virus budding takes place (16, 30), and mutants that lack UL11 are defective for the production of virions, resulting in an increased number of unenveloped capsids in the cytoplasm (5, 9, 19). The UL11-UL16 interaction has since been confirmed by other groups (15, 37), and more recently, we have found that the interaction is direct and requires free cysteines present within UL16 (41). That is, chemical modification of free cysteines in UL16 with N-ethylmaleimide (NEM) blocks the interaction with UL11. On the UL11 side of the interaction, LI and acidic cluster motifs are needed for binding (17, 41). UL16 is a 373-amino-acid protein that is also conserved among herpesviruses and exhibits dynamic capsid-binding properties. Although it is found in both the cytoplasm and the nucleus of the infected cell, it is only stably associated with capsids isolated from the cytoplasm (20, 24, 26). This finding, combined with the ability of UL11 to accumulate at the site of budding, led us to hypothesize that the UL11-UL16 interaction provides a bridging function to assist the capsid in acquiring its envelope (17). However, sometime after budding—as the virus egresses from the cell—the interaction of UL16 with the capsid is destabilized (20). And, as mentioned earlier, binding of the virion to its attachment receptors on the host cell surface (heparan sulfate) further disrupts the association of UL16 with the capsid (21). Free cysteines appear to play a critical role in this outside-in signaling event, because treatment of extracellular virions with NEM prior to cell binding prevents the release of UL16 from the capsid (21). While UL16 was the most abundant protein pulled out of infected cell lysates in our search for UL11 binding partners, a much less prominent, but highly reproducible, ∼65-kDa species was also observed (17). Like UL16, this unknown protein was absent when either the LI or acidic cluster motifs were eliminated from the glutathione S-transferase (GST)-UL11 construct used in the experiment. This suggested that the unknown protein was obtained by either (i) competing with UL16 for binding to the same motifs within UL11 or (ii) binding to UL11 indirectly through an interaction with UL16. Because the LI and acidic cluster motifs of UL11 are recognized by host proteins for trafficking through lipid rafts (6, 16), the first hypothesis seemed likely; however, because UL16 participates in a complex signaling pathway within the virion, it was possible that the unknown protein would be a virus-encoded component. The purpose of the experiments described in this report was to identify this unknown protein and to determine how it fits into the UL16 network of interactions.

All herpesviruses encode a homolog of the herpes simplex virus type-1 UL11 tegument protein. Deletion of UL11 disrupts virus envelopment, causes capsid accumulation within the cytoplasm, and reduces virus release. UL11 requires acylation with myristate and palmitate for membrane binding, lipid raft trafficking, and accumulation at the site of virus envelopment. Thus, it was predicted that acylation of UL11 would be necessary for efficient virion production, similar to HIV-1 Gag which requires myristylation for virus production. Accordingly, recombinant viruses were created to express UL11 derivatives that are not acylated, are partially acylated, or contain foreign acylation signals. Unexpectedly, the non-acylated UL11 rescued some growth defects of a UL11-null mutant, even though the unmodified protein was unstable. Furthermore, a myristylated and palmitylated chimera did not fully rescue the null virus. These results suggest that UL11 maintains some function(s) when not membrane-bound, and the sequence context of the acylations is important for UL11 function.

The UL11 and UL16 tegument proteins of herpes simplex virus are conserved throughout the herpesvirus family. Previous studies have shown that these proteins interact, perhaps to link UL16-bound nucleocapsids to UL11, which resides on the cytoplasmic face of the trans -Golgi network, where maturation budding occurs. Little is known about the interaction except that it requires the leucine-isoleucine (LI) and acidic cluster motifs in UL11 and that no other viral proteins are involved. In particular, the important question of whether these two proteins bind to each other directly has not been addressed. Accordingly, UL11 and UL16 were expressed in bacteria, and the purified proteins were found to retain the ability to interact in a manner that was dependent upon the LI and acidic cluster. In an attempt to map the UL11-binding site contained in UL16, a large number of deletion mutants were constructed. The first 40 (nonconserved) amino acids were found to be dispensable, but all the other constructs failed to bind UL11 or had poor expression in transfected cells, suggesting that UL16 is very sensitive to alterations and probably lacks a multidomain structure. As an alternative strategy for identifying residues that are important for the interaction, the cysteines of UL16 were investigated, because many of these are highly conserved. Approximately half of the 20 cysteines in UL16 have been shown to be covalently modified by N -ethylmaleimide, and this treatment was found to block the interaction with UL11. Moreover, individual serine replacements of six of the most conserved cysteine residues were made, and four of these disrupted the interaction with UL11 without affecting protein stability. However, the UL11-UL16 interaction does not involve the formation of interspecies disulfide bonds, because binding occurred even when all the cysteines in UL11 were eliminated. Thus, UL16 directly interacts with UL11 and does so in a manner that requires free cysteines.

We have made the surprising discovery that the interactions of herpes simplex virus with its initial cell attachment receptor induce a rapid and highly efficient structural change in the tegument, the region of the virion situated between the membrane and the capsid. It has been known for nearly a decade that viruses can trigger host signaling pathways when they bind to receptors on the cell surface; however, until now there has been no evidence that a signal can be sent in reverse—from the “outside in”—across a viral membrane. Evidence for this signaling event was found during studies of UL16, a tegument protein that is conserved among all the herpesviruses. Previous work has demonstrated that UL16 is bound to capsids isolated from the cytoplasm of infected cells, but this interaction is destabilized during subsequent egress steps, leading to release of the extracellular virion. Pretreatment with N -ethylmaleimide, a small, membrane-permeating compound that covalently modifies free cysteines, restabilizes the interaction, thereby permitting the capsid-UL16 complex to be isolated following disruption of virions with NP-40. In the experiments described here, we found that the natural signal for release of UL16 from capsids is sent when virions merely bind to cells at 4°C. The internal change was also observed upon binding to immobilized heparin in a manner that requires viral glycoprotein C. This represents the first example of signaling across a viral envelope following receptor binding.