Singapore's Total Diet Study (2021-2023): Study Design, Methodology, and Relevance to Ensuring Food Safety.

A total diet study is often used to evaluate a population's baseline dietary exposure to chemical hazards from across the diet. In 2021-2023, Singapore carried out a TDS, and this article presents an overview of the study design and methodological selections in Singapore's TDS, as well as its relevance to ensuring food safety. A food consumption survey was conducted on Singapore citizens and permanent residents, where food consumption patterns of the Singapore population were identified. The selection of chemical hazards and foods for inclusion in Singapore's TDS, as well as principal considerations on sampling, food preparation, and analytical testing are discussed. Commonly consumed foods by the Singapore population in food categories such as grain and grain-based products, meat and meat products, fish and seafood, vegetables, fruits, milk and dairy products were included in this study, and mean concentrations of chemicals tested in each food category were reported, with food categories possessing higher levels identified. Future work will include dietary exposure assessments for the population and analysis of the contributions by food and cooking method.

The detection, characterization, and quantification of dominant degradation products of nisin A and Z in selected dairy products by liquid chromatography-high-resolution mass spectrometry technique.

Nisin, a bacteriocin produced through fermentation using bacterium Lactococcus lactis, has several commercial variants such as nisin A and nisin Z. Nisin serves as a natural preservative with antimicrobial properties in various food products, including dairy and beverages, for extending product shelf life. The efficacy and safety of nisin A as a bacteriocin has been well characterized. However, there is limited evidence regarding the efficacy, stability, and safety of nisin Z as a food preservative, as it has not undergone comprehensive regulatory reviews. In this work, we studied the stability of nisin A and Z in a selection of yogurt drinks and found nisin to be unstable, particularly in fruit-flavored yogurt drinks. Both nisin A and Z could experience significant degradation leading to the nisin parent ion peaks dropping below detectable level before the product's expiry date. Compared with nisin A, the formation of oxidized metabolite nisin Z+O appeared to be the predominant reaction for nisin Z. These findings highlight the need for further scientific research to understand the behavior of nisin Z under different application conditions, which is crucial for assessing the efficacy and safety of nisin Z under these conditions. One potential application of this knowledge is to optimize the formulation of yogurt-based drinks to stabilize nisin Z and sustain its biopreservative function throughout the product's shelf life. Additionally, the current study shows that for the testing of the presence of nisin A or nisin Z, it is imperative to cover both the parent and the main degradant(s) of nisin. This is especially true for nisin Z, for which the regulatory approval status may vary in different markets. As such, the confirmative identification of nisin Z and its key metabolites in commercial products would be essential.

Identification and Characterization of Mercury Contamination in Vegetables and Herbs Cultivated on a Commercial Vertical Indoor Farming System with Light-Emitting Diode Lighting: Unveiling an Unusual Food Safety Risk of Some Improperly Manufactured High-Density Agricultural Production Systems.

Artificial grow lights, such as light-emitting diodes (LEDs) and fluorescent grow lights, are commonly used in modern day indoor farming, citing advantages in energy efficiency and a higher controlled environment. However, the use of LEDs poses a risk in mercury contaminations as a result of its production process, specifically LEDs with polyurethane encapsulates that were traditionally produced using mercury resins as a catalyst. A total of 10.0 ppm of mercury was detected in a curly kale sample harvested from an indoor hydroponic vegetable farm, exceeding Singapore Food Regulation's limit of 0.05 ppm. Vegetables, farming inputs, and surface swabs from the affected farm were analyzed using wet acid digestion followed by cold vapor atomic absorption spectroscopy analysis. The investigation found high concentrations of mercury in the LED encapsulant, and the encapsulant material was identified to be polyurethane by Fourier transform infrared spectroscopy and pyrolysis-gas chromatography-mass spectrometry analysis, indicating the source of mercury contamination to be the LED polyurethane encapsulant.

Spatial distribution of elements during osteoarthritis disease progression using synchrotron X-ray fluorescence microscopy.

The osteochondral interface is a thin layer that connects hyaline cartilage to subchondral bone. Subcellular elemental distribution can be visualised using synchrotron X-ray fluorescence microscopy (SR-XFM) (1 µm). This study aims to determine the relationship between elemental distribution and osteoarthritis (OA) progression based on disease severity. Using modified Mankin scores, we collected tibia plates from 9 knee OA patients who underwent knee replacement surgery and graded them as intact cartilage (non-OA) or degraded cartilage (OA). We used a tape-assisted system with a silicon nitride sandwich structure to collect fresh-frozen osteochondral sections, and changes in the osteochondral unit were defined using quantified SR-XFM elemental mapping at the Australian synchrotron's XFM beamline. Non-OA osteochondral samples were found to have significantly different zinc (Zn) and calcium (Ca) compositions than OA samples. The tidemark separating noncalcified and calcified cartilage was rich in zinc. Zn levels in OA samples were lower than in non-OA samples (P = 0.0072). In OA samples, the tidemark had less Ca than the calcified cartilage zone and subchondral bone plate (P < 0.0001). The Zn-strontium (Sr) colocalisation index was higher in OA samples than in non-OA samples. The lead, potassium, phosphate, sulphur, and chloride distributions were not significantly different (P > 0.05). In conclusion, SR-XFM analysis revealed spatial elemental distribution at the subcellular level during OA development.

A technique for preparing undecalcified osteochondral fresh frozen sections for elemental mapping and understanding disease etiology.

The anatomy of the osteochondral junction is complex because several tissue components exist as a unit, including uncalcified cartilage (with superficial, middle, and deep layers), calcified cartilage, and subchondral bone. Furthermore, it is difficult to study because this region is made up of a variety of cell types and extracellular matrix compositions. Using X-ray fluorescence microscopy, we present a protocol for simultaneous elemental detection on fresh frozen samples. We transferred the osteochondral sample using a tape-assisted system and successfully tested it in synchrotron X-ray fluorescence. This protocol elucidates the distinct distribution of elements at the human knee's osteochondral junction, making it a useful tool for analyzing the co-distribution of various elements in both healthy and diseased states.

Impaired signaling for neuromuscular synaptic maintenance is a feature of Motor Neuron Disease.

A central event in the pathogenesis of motor neuron disease (MND) is the loss of neuromuscular junctions (NMJs), yet the mechanisms that lead to this event in MND remain to be fully elucidated. Maintenance of the NMJ relies upon neural agrin (n-agrin) which, when released from the nerve terminal, activates the postsynaptic Muscle Specific Kinase (MuSK) signaling complex to stabilize clusters of acetylcholine receptors. Here, we report that muscle from MND patients has an increased proportion of slow fibers and muscle fibers with smaller diameter. Muscle cells cultured from MND biopsies failed to form large clusters of acetylcholine receptors in response to either non-MND human motor axons or n-agrin. Furthermore, levels of expression of MuSK, and MuSK-complex components: LRP4, Caveolin-3, and Dok7 differed between muscle cells cultured from MND patients compared to those from non-MND controls. To our knowledge, this is the first time a fault in the n-agrin-LRP4-MuSK signaling pathway has been identified in muscle from MND patients. Our results highlight the n-agrin-LRP4-MuSK signaling pathway as a potential therapeutic target to prolong muscle function in MND.

Seizures Are Associated with Blood-Brain Barrier Disruption in a Piglet Model of Neonatal Hypoxic-Ischaemic Encephalopathy.

Seizures in the neonatal period are most often symptomatic of central nervous system (CNS) dysfunction and the most common cause is hypoxic-ischaemic encephalopathy (HIE). Seizures are associated with poor long-term outcomes and increased neuropathology. Blood-brain barrier (BBB) disruption and inflammation may contribute to seizures and increased neuropathology but are incompletely understood in neonatal HIE. The aim of this study was to investigate the impact of seizures on BBB integrity in a preclinical model of neonatal hypoxic-ischaemic (HI) injury. Piglets (age: <24 h) were subjected to a 30-min HI insult followed by recovery to 72 h post-insult. Amplitude-integrated electroencephalography (aEEG) was performed and seizure burden and background aEEG pattern were analysed. BBB disruption was evaluated in the parietal cortex and hippocampus by means of immunohistochemistry and Western blot. mRNA and protein expression of tight-junction proteins (zonula-occludens 1 [ZO1], occludin [OCLN], and claudin-5 [CLDN5]) was assessed using quantitative polymerase chain reaction (qPCR) and Western blot. In addition, mRNA from genes associated with BBB disruption vascular endothelial growth factor (VEGF) and matrix metalloproteinase 2 (MMP2) as well as inflammatory cytokines and chemokines was assessed with qPCR. Piglets that developed seizures following HI (HI-Sz) had significantly greater injury, as demonstrated by poorer aEEG background pattern scores, lower neurobehavioural scores, and greater histopathology. HI-Sz animals had severe IgG extravasation into brain tissue and uptake into neurons as well as significantly greater levels of IgG in both brain regions as assessed by Western blot. IgG protein in both brain regions was significantly associated with seizure burden, aEEG pattern scores, and neurobehavioural scores. There was no difference in mRNA expression of the tight junctions, however a significant loss of ZO1 and OCLN protein was observed in the parietal cortex. The inflammatory genes TGFß, IL1ß, IL8, IL6, and TNFα were significantly upregulated in HI-Sz animals. MMP2 was significantly increased in animals with seizures compared with animals without seizures. Increasing our understanding of neuropathology associated with seizure is vital because of the association between seizure and poor outcomes. Investigating the BBB is a major untapped area of research and a potential avenue for novel treatments.

Ibuprofen Treatment Reduces the Neuroinflammatory Response and Associated Neuronal and White Matter Impairment in the Growth Restricted Newborn.

Intrauterine growth restriction (IUGR) is a condition where the fetus does not achieve optimal growth, commonly caused by placental insufficiency. The chronic decrease in blood flow restricts oxygen and nutrient supply to the fetus, which can damage numerous organ systems, with the fetal brain being particularly vulnerable. Although white matter and neuronal injury are evident in IUGR infants, the specific mechanisms underlying these changes are poorly understood. Inflammation is considered to be a main driver in exacerbating brain injury. Using a spontaneous piglet model of IUGR, we aim to determine whether administration of the anti-inflammatory drug ibuprofen will decrease inflammation at postnatal day 4 (P4). The treatment group received ibuprofen (20 mg/kg/day on day 1 and 10 mg/kg/day on days 2 and 3) in piglet formula during the morning feed each day and brains examined on P4. Markers of inflammation, apoptosis, cell proliferation, neuronal injury, and white matter injury were examined. Ibuprofen treatment ameliorated the increase in numbers of microglia and astrocytes in the parietal cortex and white matter tracts of the IUGR piglet brain on P4 as well as decreasing proinflammatory cytokines. Ibuprofen treatment prevented the reduction in apoptosis, neuronal cell counts, and myelin index in the IUGR piglets. Our findings demonstrate ibuprofen reduces the inflammatory response in the IUGR neonatal brain and concurrently reduces neuronal and white matter impairment.

Neuropathology in intrauterine growth restricted newborn piglets is associated with glial activation and proinflammatory status in the brain.

BACKGROUND: The fetal brain is particularly vulnerable to intrauterine growth restriction (IUGR) conditions evidenced by neuronal and white matter abnormalities and altered neurodevelopment in the IUGR infant. To further our understanding of neurodevelopment in the newborn IUGR brain, clinically relevant models of IUGR are required. This information is critical for the design and implementation of successful therapeutic interventions to reduce aberrant brain development in the IUGR newborn. We utilise the piglet as a model of IUGR as growth restriction occurs spontaneously in the pig as a result of placental insufficiency, making it a highly relevant model of human IUGR. The purpose of this study was to characterise neuropathology and neuroinflammation in the neonatal IUGR piglet brain. METHODS: Newborn IUGR (< 5th centile) and normally grown (NG) piglets were euthanased on postnatal day 1 (P1; < 18 h) or P4. Immunohistochemistry was utilised to examine neuronal, white matter and inflammatory responses, and PCR for cytokine analysis in parietal cortex of IUGR and NG piglets. RESULTS: The IUGR piglet brain displayed less NeuN-positive cells and reduced myelination at both P1 and P4 in the parietal cortex, indicating neuronal and white matter disruption. A concurrent decrease in Ki67-positive proliferative cells and increase in cell death (caspase-3) in the IUGR piglet brain was also apparent on P4. We observed significant increases in the number of both Iba-1-positive microglia and GFAP-positive astrocytes in the white matter in IUGR piglet brain on both P1 and P4 compared with NG piglets. These increases were associated with a change in activation state, as noted by altered glial morphology. This inflammatory state was further evident with increased expression levels of proinflammatory cytokines (interleukin-1ß, tumour necrosis factor-α) and decreased levels of anti-inflammatory cytokines (interleukin-4 and -10) observed in the IUGR piglet brains. CONCLUSIONS: These findings suggest that the piglet model of IUGR displays the characteristic neuropathological outcomes of neuronal and white matter impairment similar to those reported in the IUGR human brain. The activated glial morphology and elevated proinflammatory cytokines is indicative of an inflammatory response that may be associated with neuronal damage and white matter disruption. These findings support the use of the piglet as a pre-clinical model for studying mechanisms of altered neurodevelopment in the IUGR newborn.

Defects in synaptic transmission at the neuromuscular junction precede motor deficits in a TDP-43Q331K transgenic mouse model of amyotrophic lateral sclerosis.

Transactive response DNA-binding protein-43 (TDP-43) is involved in gene regulation via the control of RNA transcription, splicing, and transport. TDP-43 is a major protein component of ubiquinated inclusions that are found in amyotrophic lateral sclerosis (ALS); however, the function of TDP-43 at the neuromuscular junction (NMJ) and its role in ALS pathogenesis is largely unknown. Here, we show that TDP-43Q331K mutation in mice resulted in impaired neurotransmission by age 3 mo, preceding deficits in motor function and motor neuron loss, which were observed from age 10 mo. These defects were in the effective fusion and release of synaptic vesicles within the motor nerve terminal and manifested in decreased quantal content and reduced probability of quantal release. We observed morphologic alterations that were associated with the TDP-43Q331K mutation, such as aberrant innervation patterns and the distribution of synaptic vesicle-related proteins, which is indicative of a failing NMJ undergoing synaptic remodeling. These findings support a growing acceptance that dysregulation of the NMJ function is a key early event in the pathology of ALS.-Chand, K. K., Lee, K. M., Lee, J. D., Qiu, H., Willis, E. F., Lavidis, N. A., Hilliard, M. A., Noakes, P. G. Defects in synaptic transmission at the neuromuscular junction precede motor deficits in a TDP-43Q331K transgenic mouse model of amyotrophic lateral sclerosis.

Complement C5a-C5aR1 signalling drives skeletal muscle macrophage recruitment in the hSOD1G93A mouse model of amyotrophic lateral sclerosis.

BACKGROUND: The terminal pathway of the innate immune complement system is implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). Terminal complement activation leads to generation of C5a, which through its receptor, C5aR1, drives immune cell recruitment and activation. Importantly, genetic or pharmacological blockage of C5aR1 improves motor performance and reduces disease pathology in hSOD1G93A rodent models of ALS. In this study, we aimed to explore the potential mechanisms of C5aR1-mediated pathology in hSOD1G93A mice by examining their skeletal muscles. RESULTS: We found elevated levels of C1qB, C4, fB, C3, C5a, and C5aR1 in tibialis anterior muscles of hSOD1G93A mice, which increased with disease progression. Macrophage cell numbers also progressively increased in hSOD1G93A muscles in line with disease progression. Immuno-localisation demonstrated that C5aR1 was expressed predominantly on macrophages within hSOD1G93A skeletal muscles. Notably, hSOD1G93A × C5aR1-/- mice showed markedly decreased numbers of infiltrating macrophages, along with reduced neuromuscular denervation and improved grip strength in hind limb skeletal muscles, when compared to hSOD1G93A mice. CONCLUSION: These results indicate that terminal complement activation and C5a production occur in skeletal muscle tissue of hSOD1G93A mice, and that C5a-C5aR1 signalling contributes to the recruitment of macrophages that may accelerate muscle denervation in these ALS mice.

Functional decline at the aging neuromuscular junction is associated with altered laminin-α4 expression.

Laminin-α4 is involved in the alignment of active zones to postjunctional folds at the neuromuscular junction (NMJ). Prior study has implicated laminin-α4 in NMJ maintenance, with altered NMJ morphology observed in adult laminin-α4 deficient mice (lama4-/-). The present study further investigated the role of laminin-α4 in NMJ maintenance by functional characterization of transmission properties, morphological investigation of synaptic proteins including synaptic laminin-α4, and neuromotor behavioral testing. Results showed maintained perturbed transmission properties at lama4-/- NMJs from adult (3 months) through to aged (18-22 months). Hind-limb grip force demonstrated similar trends as transmission properties, with maintained weaker grip force across age groups in lama4-/-. Interestingly, both transmission properties and hind-limb grip force in aged wild-types resembled those observed in adult lama4-/-. Most significantly, altered expression of laminin-α4 was noted at the wild-type NMJs prior to the observed decline in transmission properties, suggesting that altered laminin-α4 expression precedes the decline of neurotransmission in aging wild-types. These findings significantly support the role of laminin-α4 in maintenance of the NMJ during aging.

Loss of laminin-α4 results in pre- and postsynaptic modifications at the neuromuscular junction.

Synaptic basal lamina such as laminin-421 (α4ß2γ1) mediate differentiation of the neuromuscular junction (NMJ). Laminins interact with their pre- or postsynaptic receptors to provide stability and alignment of the pre- to postsynaptic specializations. Knockout of the laminin-α4 gene (lama4) does not alter gross NMJ morphogenesis. However, mice deficient in laminin-α4 (lama4-/- mice) display disruptions in the alignment of the active zones and postsynaptic folds at the NMJ, although the physiological consequences of this loss have not been examined. The present study investigated the differences in neurotransmission during the early development and maturation of the NMJ in lama4-/- and wild-type mice. Lama4-/- NMJs demonstrated a decrease in miniature end-plate potential (EPP) frequency and increased amplitude of miniature EPPs and evoked EPPs. Binomial parameters analysis of neurotransmitter release revealed a decrease in quantal release, the result of a decrease in the number of active release sites, but not in the probability of transmitter release. Lama4-/- NMJs displayed higher levels of synaptic depression under high-frequency stimulation and altered facilitation, suggesting compromised delivery of synaptic vesicles. This idea is supported by our molecular investigations of lama4-/- NMJs, where we see altered distribution of Bassoon, a molecular component of active zones, presumably resulting from perturbed neurotransmission.-Chand, K. K., Lee, K. M., Lavidis, N. A., Noakes, P. G. Loss of laminin-α4 results in pre- and postsynaptic modifications at the neuromuscular junction.

Tick holocyclotoxins trigger host paralysis by presynaptic inhibition.

Ticks are important vectors of pathogens and secreted neurotoxins with approximately 69 out of 692 tick species having the ability to induce severe toxicoses in their hosts. The Australian paralysis tick (Ixodes holocyclus) is known to be one of the most virulent tick species producing a flaccid paralysis and fatalities caused by a family of neurotoxins known as holocyclotoxins (HTs). The paralysis mechanism of these toxins is temperature dependent and is thought to involve inhibition of acetylcholine levels at the neuromuscular junction. However, the target and mechanism of this inhibition remain uncharacterised. Here, we report that three members of the holocyclotoxin family; HT-1 (GenBank AY766147), HT-3 (GenBank KP096303) and HT-12 (GenBank KP963967) induce muscle paralysis by inhibiting the dependence of transmitter release on extracellular calcium. Previous study was conducted using extracts from tick salivary glands, while the present study is the first to use pure toxins from I. holocyclus. Our findings provide greater insight into the mechanisms by which these toxins act to induce paralysis.

Loss of ß2-laminin alters calcium sensitivity and voltage-gated calcium channel maturation of neurotransmission at the neuromuscular junction.

KEY POINTS: Neuromuscular junctions from ß2-laminin-deficient mice exhibit lower levels of calcium sensitivity. Loss of ß2-laminin leads to a failure in switching from N- to P/Q-type voltage-gated calcium channel (VGCC)-mediated transmitter release that normally occurs with neuromuscular junction maturation. The motor nerve terminals from ß2-laminin-deficient mice fail to up-regulate the expression of P/Q-type VGCCs clusters and down-regulate N-type VGCCs clusters, as they mature. There is decreased co-localisation of presynaptic specialisations in ß2-laminin-deficient neuromuscular junctions as a consequence of lesser P/Q-type VGCC expression. These findings support the idea that ß2-laminin is critical in the organisation and maintenance of active zones at the neuromuscular junction via its interaction with P/Q-type VGCCs, which aid in stabilisation of the synapse. ß2-laminin is a key mediator in the differentiation and formation of the skeletal neuromuscular junction. Loss of ß2-laminin results in significant structural and functional aberrations such as decreased number of active zones and reduced spontaneous release of transmitter. In vitro ß2-laminin has been shown to bind directly to the pore forming subunit of P/Q-type voltage-gated calcium channels (VGCCs). Neurotransmission is initially mediated by N-type VGCCs, but by postnatal day 18 switches to P/Q-type VGCC dominance. The present study investigated the changes in neurotransmission during the switch from N- to P/Q-type VGCC-mediated transmitter release at ß2-laminin-deficient junctions. Analysis of the relationship between quantal content and extracellular calcium concentrations demonstrated a decrease in the calcium sensitivity, but no change in calcium dependence at ß2-laminin-deficient junctions. Electrophysiological studies on VGCC sub-types involved in transmitter release indicate N-type VGCCs remain the primary mediator of transmitter release at matured ß2-laminin-deficient junctions. Immunohistochemical analyses displayed irregularly shaped and immature ß2-laminin-deficient neuromuscular junctions when compared to matured wild-type junctions. ß2-laminin-deficient junctions also maintained the presence of N-type VGCC clustering within the presynaptic membrane, which supported the functional findings of the present study. We conclude that ß2-laminin is a key regulator in development of the NMJ, with its loss resulting in reduced transmitter release due to decreased calcium sensitivity stemming from a failure to switch from N- to P/Q-type VGCC-mediated synaptic transmission.