Coordinated signaling among neurons, glia, and the vasculature is essential for nervous system development. In the developing retina, spontaneous cholinergic retinal waves are the primary source of neural activity during the early maturation of the vasculature. Here, we test the hypothesis that retinal waves influence angiogenesis and the maturation of the glial-vascular interface. We first found that retinal vasculature grew normally in mice lacking β2-containing nicotinic acetylcholine receptor-mediated retinal waves, demonstrating that early spontaneous activity is not required for angiogenesis. We next examined how Müller glia establish and signal at the developing glial-vascular interface. Sparse labeling and immunohistochemistry revealed that Müller glial lateral processes closely associate with endothelial tip cells during intermediate and deep layer angiogenesis and establish Aquaporin-4-enriched endfeet at vascular contact sites from the earliest stages of vascular growth. These associations were stable across development and persisted even when diving-vessel trajectories were disrupted in Piezo2 conditional knockouts. To determine whether glial signaling at the vascular interface is coupled to retinal waves, we combined two-photon calcium imaging with simultaneous retinal ganglion cell voltage-clamp recordings. Müller glial endfeet exhibited robust, compartmentalized calcium transients that were largely uncorrelated with retinal waves. Although blocking GABA-A receptors with gabazine increased wave-correlated activity in all glial compartments, the majority of endfoot calcium signaling remained wave-independent. Together, these findings support a model in which both angiogenesis and establishment of the Müller glial-vascular interface proceed through wave-independent developmental programs.

Understanding consumer habits and practices of cosmetics and personal care products (PCP) is essential to generate realistic product exposure data for the safety assessment of ingredients such as fragrance materials. Product usages can vary across regions due to differences in cultural norms, seasonal and climate conditions, and the availability of different product forms, yet there is limited data published on cosmetics and PCP use outside of North America and Europe. This study reports the habits and practices of cosmetics and PCP (such as frequency and amount of use) in Singapore where participants (n=494, aged 21-64 years) recorded their product usages and had their products weighed over a two-week period. Overall, similar use patterns were observed across demographic groups within the Singapore population for most of the products surveyed, as were the expected usage amounts. Additionally, the Singaporean dataset was mapped onto the Creme-RIFM aggregate exposure model to assess exposure estimates. Preliminary comparisons with product exposures observed in the United States (US) and Europe suggest that exposures in Singapore are comparable. Findings from this study will contribute to the Creme-RIFM model, expanding its geographic scope and applicability for the global safety assessment of fragrance ingredients and fragranced products.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen able to cause life- and sight-threatening infections. Once considered an extracellular pathogen, numerous studies have shown it can survive intracellularly. Previously, we showed that P. aeruginosa inside cells can diversify into distinct subpopulations in vacuoles and the cytoplasm. Here, we report that the transition from vacuoles to cytoplasm requires collaboration with the extracellular subpopulation, through Ca2+ influx enabled by their type III secretion system (T3SS) translocon pore proteins. Moreover, we show that collaboration among P. aeruginosa subpopulations can contribute to disseminating intracellular bacteria in vivo in a mouse infection model. This study lays the groundwork for future investigations into how cooperation between extracellular and intracellular bacteria within the host contributes to disease progression and persistence.

Risk analysis is an internationally recognized process adopted by food regulatory bodies across the world. It has three parts—risk assessment, risk management, and risk communication. This article briefly explains the principles underlying risk analysis as applied to food safety, with an emphasis on risk assessment, the scientific methodology used to identify, evaluate and prioritize potential risks to human health associated with food products and processes. Key steps taken to conduct a food safety risk assessment are explained along with an illustrative example of how to conduct a risk assessment for chemical hazards. Overall, food safety risk analysis is an important tool for protecting public health by identifying and mitigating potential food safety risks.

Most dietary intake estimates used in the regulatory risk assessment of flavouring substances assume that the flavouring substance is present in all foods at a given concentration. They do not account for the use of alternate flavouring substances for the same taste modality, nor do they consider the use of differing concentrations within a particular food category. This highly conservative assumption leads to an over-estimation of exposure. To refine this assumption in exposure models, the probability of addition can be utilised. In this study, a methodology to estimate the probability of addition was developed, using the volume of a flavouring substance sold for flavours created for a particular food category against its total volume sold for all flavouring use. The method was trialled, as a proof of principle, using historical data sets of two test flavouring substances, benzaldehyde (a large volume/use material) and bornyl acetate (a low volume/use material), and the impact on dietary exposure assessment was assessed. The resulting exposure estimates for both flavouring substances were shown to be significantly lower than those without the incorporation of probability of addition, demonstrating that current methods are overly conservative and that the use of probability of addition provides a more realistic estimate of exposure of flavouring substances present in foods. The current assumption for consumer exposure is that a flavouring ingredient is present at its maximum allowed level in all foods. Using historical volume sales data, we demonstrate how likely it is that a consumer will actually be exposed to a flavouring ingredient by applying the probability of addition.image

Engineered nanoparticles (NPs) are extensively used in the food industry, yet safety concerns remain. The lack of validated methodologies is a bottleneck towards resolving this uncertainty. Hence, the current study aims to compare two cell models by examining the toxicological impacts of two food-relevant NPs (SiO2 and Ag) on intestinal epithelia using monolayer Caco-2 cells and full-thickness 3D tissue models of human small intestines (EpiIntestinal™). Comprehensive characterization and dosimetric analysis of the NPs were performed to determine effective doses and model realistic exposures. Neither genotoxicity nor cytotoxicity were detected in the 3D tissues after NP treatment, while the 2D cultures exhibited cytotoxic response from Ag NP treatment for 24 h at 1 μg/ml. Hyperspectral imaging and transmission electron microscopy confirmed uptake of both NPs by cells in both 2D and 3D culture models. Ag NPs caused an increase in autophagy, whereas SiO2 NPs induced increased cytoplasmic vacuolization. Based on realistic exposure levels studied, the 3D small intestinal tissue model was found to be more resilient to NP treatment compared to 2D cell monolayers. This comparative approach towards toxicological assessment of food relevant NPs could be used as a framework for future analysis of NP behavior and nanotoxicity in the gut.

While valuable for hazard identification, in vitro genotoxicity tests that are conducted in 2D monolayer cell cultures possess limitations to accurately envisage in vivo outcomes and may result in unnecessary follow-up in vivo studies. One limitation is their relevance to actual human exposure and the assessment of genotoxicity in tissues representative of “sites-of-first-contact” with genotoxic agents. The revised OECD testing guidelines had emphasized the need for considering site-of-first-contact effect and should be included when carrying out genotoxic hazard investigations. Till date, only in vivo studies can adequately address the oral/gastrointestinal route of exposure and there exists no validated in vitro assay for the gut for ingestible materials. Previously, we described the development of a new human Reconstructed Intestine Micronuclei Cytome (RICyt) assay using EpiIntestinal™ microtissues for site-of-contact genotoxic hazard identification of orally ingested materials. The human small intestine is where approximately 90% of the digestion and adsorption of food occurs and a major exposure site of ingested genotoxicants, thus a key site for evaluation. Here, we have conducted an investigation on the predictive performance of the RICyt assay. The assay platform was challenged with a training set of 16 test materials which include standard reference genotoxins and non-genotoxins as well as food relevant substances with various mode of actions. The overall RICyt accuracy was 92.9% with sensitivity of 83.3% and specificity of 100%. Majority of the test materials were correctly identified during this within-laboratory investigation though yielded one false negative and two equivocal outcomes. These results emphasize that the RICyt assay in EpiIntestinal™ microtissues has adequate predictivity and is a promising in vitro tool for genotoxic hazard identification of orally ingested substances.

For genotoxic carcinogens, covalent binding to DNA is a critical initiating event in tumorigenesis. The present research investigated dose-effect relationships of three genotoxic carcinogens representing different structural classes, 2-acetylaminofluorene (2-AAF), benzo[a]pyrene (B[a]P) and quinoline (QUI), to assess the existence of no-observed-effect-levels (NOELs) for the formation of DNA adducts. Carcinogens were administered into the air sac of fertilized turkey eggs over wide dose ranges in three daily injections on days 22 to 24 of incubation. DNA adducts were measured in the fetal turkey livers by the 32P-nucleotide postlabeling (NPL) assay. B[a]P and QUI produced DNA adducts in a dosage-related manner and exhibited NOELs at 0.65 and 0.35 mg/kg bw/day, respectively. In contrast, 2-AAF formed DNA adducts at all tested dosages down to 0.005 mg/kg bw/day. Benchmark dose (BMD) analysis identified the potencies of 2-AAF and QUI to be similar, while B[a]P was the least potent compound. Overall, findings in fetal turkey livers demonstrated that exposure levels to genotoxic compounds that do not result in DNA adducts can exist but are not evident with all carcinogens of this type. The use of mechanistic dose-effect studies for genotoxic endpoints can provide critical information for prioritization of concerns for risk assessment.

Bone loss, commonly seen in osteoporosis, is a condition that entails a progressive decline of bone mineral density and microarchitecture, often seen in post-menopausal women. Bone loss has also been widely reported in astronauts exposed to a plethora of stressors and in patients with osteoporosis following radiotherapy for cancer. Studies on mechanisms are well documented but the causal connectivity of events to bone loss development remains incompletely understood. Herein, the adverse outcome pathway (AOP) framework was used to organize data and develop a qualitative AOP beginning from deposition of energy (the molecular initiating event) to bone loss (the adverse outcome). This qualitative AOP was developed in collaboration with bone loss research experts to aggregate relevant findings, supporting ongoing efforts to understand and mitigate human system risks associated with radiation exposures. A literature review was conducted to compile and evaluate the state of knowledge based on the modified Bradford Hill criteria. Following review of 2029 studies, an empirically supported AOP was developed, showing the progression to bone loss through many factors affecting the activities of bone-forming osteoblasts and bone-resorbing osteoclasts. The structural, functional, and quantitative basis of each proposed relationship was defined, for inference of causal changes between key events. Current knowledge and its gaps relating to dose-, time- and incidence-concordance across the key events were identified, as well as modulating factors that influence linkages. The new priorities for research informed by the AOP highlight areas for improvement to enable development of a quantitative AOP used to support risk assessment strategies for space travel or cancer radiotherapy.

The consumption of sugar and non-nutritive sweeteners has been associated with poor health outcomes. The aim of this paper was to provide a comparison of the range of sweetened or flavoured beverages between two high-income countries in the Asia-Pacific region: Australia and Singapore. Following the FoodTrackTM methodology, nutrition, labelling, and price data were collected from major Australian and Singaporean supermarket chains and convenience stores. The nutrient profiles of products were tested for differences using Kruskal–Wallis and Mann–Whitney U tests. The greatest number of products collected in Australia were from the ‘carbonated beverages’ category (n = 215, 40%), and in Singapore the greatest number of products were from the ‘tea and coffee ready-to-drink’ category (n = 182, 35%). There were more calorically sweetened beverages in Singapore compared with Australia (n = 462/517 vs. n = 374/531, p < 0.001). For calorically sweetened products, the median energy of Singaporean products was significantly higher than Australian products (134 kJ vs. 120 kJ per 100 mL, p = 0.009). In Australia, 52% of sweetened or flavoured beverages displayed a front-of-pack nutrient signposting logo, compared with 34% of sweetened or flavoured beverages in Singapore. These findings also indicate that the consumption of just one serving of calorically sweetened carbonated beverages or energy drinks would exceed the WHO maximum daily free sugar recommendations.

To maintain a stable and clear image of the world, our eyes reflexively follow the direction in which a visual scene is moving. Such gaze-stabilization mechanisms reduce image blur as we move in the environment. In non-primate mammals, this behaviour is initiated by retinal output neurons called ON-type direction-selective ganglion cells (ON-DSGCs), which detect the direction of image motion and transmit signals to brainstem nuclei that drive compensatory eye movements 1 . However, ON-DSGCs have not yet been identified in the retina of primates, raising the possibility that this reflex is mediated by cortical visual areas. Here we mined single-cell RNA transcriptomic data from primate retina to identify a candidate ON-DSGC. We then combined two-photon calcium imaging, molecular identification and morphological analysis to reveal a population of ON-DSGCs in the macaque retina. The morphology, molecular signature and GABA (γ-aminobutyric acid)-dependent mechanisms that underlie direction selectivity in primate ON-DSGCs are highly conserved with those in other mammals. We further identify a candidate ON-DSGC in human retina. The presence of ON-DSGCs in primates highlights the need to examine the contribution of subcortical retinal mechanisms to normal and aberrant gaze stabilization in the developing and mature visual system.

Understanding trends in habits and practices of cosmetics and personal care products is essential for the assessment of product exposure and simultaneously, the establishment of safe use levels for incorporated ingredients. To date, most exposure data is limited to European and USA populations, with data on Asian-specific habits and practices lacking. This data gap needs to be filled as evidence has shown that there is a large variation in product use behaviors between consumers, across and within populations. Moreover, there is a need to seek a more efficient approach of data collection. Conventional methods of collecting habits and practices data require extensive effort and are generally cost and time intensive. In this publication, we demonstrate the feasibility of employing a rapid and cost-effective online survey approach to gather habits and practices for Southeast Asia, specifically Singapore. We describe the methodology and display the type of habits and practices data that can be gathered through this approach. Although certain limitations exist, this approach can be used to effectively collect preliminary product use data across regions and different product categories.

Spontaneous activity is a hallmark of developing neural systems. In the retina, spontaneous activity comes in the form of retinal waves, comprised of three stages persisting from embryonic day 16 (E16) to eye opening at postnatal day 14 (P14). Though postnatal retinal waves have been well characterized, little is known about the spatiotemporal properties or the mechanisms mediating embryonic retinal waves, designated stage 1 waves. Using a custom-built macroscope to record spontaneous calcium transients from whole embryonic retinas, we show that stage 1 waves are initiated at several locations across the retina and propagate across a broad range of areas. Blocking gap junctions reduced the frequency and size of stage 1 waves, nearly abolishing them. Global blockade of nAChRs similarly nearly abolished stage 1 waves. Thus, stage 1 waves are mediated by a complex circuitry involving subtypes of nAChRs and gap junctions. Stage 1 waves in mice lacking the β2 subunit of the nAChRs (β2-nAChR-KO) persisted with altered propagation properties and were abolished by a gap junction blocker. To assay the impact of stage 1 waves on retinal development, we compared the spatial distribution of a subtype of retinal ganglion cells, intrinsically photosensitive retinal ganglion cells (ipRGCs), which undergo a significant amount of cell death, in WT and β2-nAChR-KO mice. We found that the developmental decrease in ipRGC density is preserved between WT and β2-nAChR-KO mice, indicating that processes regulating ipRGC numbers and distributions are not influenced by spontaneous activity.

Dairy and non-dairy (plant-based) alternatives are promoted as an essential component of a healthy diet. The purpose of this study was to evaluate the range of dairy milks and plant-based milk alternatives in supermarkets in Australia and Singapore, and to explore nutritional differences within the category, and between countries. Product information was collected in store from packaging. Products were sorted into dairy milks and plant-based milk alternatives, and further categorised as (i) breakfast drinks (12 % of products); (ii) plain milks (62 %); or (iii) flavoured milks (26 %). The nutrient profiles of products were tested for differences using Kruskal Wallis and Mann-Whitney U tests. Flavoured products contained almost double the median sugar content of plain products (8.3 g v. 4.6 g, p = 0.005). Two-thirds of the product range were dairy milks, which contained nearly four times the median saturated fat content (1.1 g v. 0.3 v, p < 0.0001) and more than double the amount of sugar (5.1 g v. 2.6 g, p < 0.0001) of plant-based milk alternatives, but three times more protein (3.3 g v. 1.0 g, p < 0.0001). Between countries, generally, calcium contents were similar across products, likely due to fortification of plant-based milk alternatives. Compared to Singapore, dairy milk and plant-based milk alternative products sold in Australia were generally higher in energy, protein and fat, but lower in carbohydrate content. Food supply differences between Singapore and Australia may be cultural and have nutritional implications.