Food structure has emerged as a critical concept with wide-ranging implications for nutrition and health. Studies with in vitro digestion models are enabling a better fundamental understanding of the structure-function relationships that define how food matrix properties change and influence nutrient release and transit during gastrointestinal digestion. Dietary lipids are of particular relevance due to their high energy density and associations with cardiometabolic risk, including postprandial rises in blood lipids. Studies using the dynamic TIM-1 digestion model enable investigations of fatty acid bioaccessibility, a precursor to lipid absorption, across a range of food products. This review presents the results of several investigations into food structure, with TIM-1 focusing on lipid digestion and instances where the findings correlate with corresponding human studies. Collectively, a better understanding of how food structure influences postprandial lipemia supports the development of food products tailored to benefit health.

Stiffening of tissue is a hallmark of cancer progression, driving invasive phenotypes through complex interactions between cells and their extracellular matrix (ECM). However, the mechanisms linking mechanical cues to ECM remodelling and invasion remain incompletely understood. Here, using an in-situ stiffening model that allows for modulation of ECM stiffness around fully formed normal mammary acini embedded in their native ECM microenvironment, we identify critical steps in basement membrane (BM) and stromal ECM remodelling during invasion and discover the molecular mechanisms driving this process. We find that stiffening of the ECM around normal mammary acini results in rapid loss and degradation of laminin (LN) and upregulation of the fibronectin (FN) secretion around the acini. This priming phase is followed by the onset of invasion which requires localized upregulation of LN production and ECM remodelling. Mechanistically, ECM production and remodelling as well as invasion is mediated by β1 integrin–FAK signalling, which activates mechanosensitive ion channels (MSCs). Further, we identify Piezo1 as the MSC downstream of β1 integrin–FAK that drives BM disruption and stromal ECM remodelling. Taken together, our results identify the mechanisms by which stiffness can trigger invasive phenotypes from normal tissues. ### Competing Interest Statement The authors have declared no competing interest.

Fundamental gaps in knowledge exist in understanding the tissue distribution of cannabinoids, cannabidiol (CBD) and tetrahydrocannabinol (THC), following oral ingestion. CBD and THC are lipid-soluble and oral bioavailability is increased when combined with long-chain fatty acid carrier oils prior to oral ingestion. Oils with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) confer positive health benefits and have yet to be examined as a carrier oil for oral cannabinoid delivery thus, examination is warranted. This study investigated the acute tissue distribution of cannabinoids in serum, adipose, brain, liver, heart, and muscle of male C57BL/6 mice at 1, 2, and 3 h (H) post oral ingestion. Mice were gavaged with CBD (5 mg/kg) and THC (1 mg/kg) combined with either sesame (SES), mixed EPA/DHA, or DHA enriched (DHA) oil as a carrier. With assistance of the Analytical Facility for Bioactive Molecules (Toronto, Canada), tissue concentration of cannabinoids was quantified using liquid chromatography with tandem mass spectrometry. SES oil resulted in a significantly greater concentration of CBD and THC (p < 0.05) across all tissues and times compared to the n-3 polyunsaturated fatty acid (PUFA) oils. The ratio of EPA:DHA in the carrier oils modestly affected distribution of cannabinoids to tissues, notably, DHA oil resulted in a greater concentration of CBD in the brain. Heart tissue had the highest concentration of CBD at 1 and 2H post-oral gavage, and adipose tissue had the highest concentration at 3H which was consistent across all three carrier oils. This study profiled the greatest number of tissues to-date for the acute distribution of CBD and THC following oral consumption with a lipid carrier in mice which demonstrated a non-uniform distribution to tissues over time. SES oil proved to be far more effective as a carrier oil at delivering orally consumed cannabinoids to tissues compared to two different n-3 PUFA containing oils. Further developing our fundamental understanding of cannabinoid distribution across tissues following their consumption from foods and pharmaceuticals are necessary to establish specific pharmacokinetic profiles to aid oral dosing strategies and maximize the bioactive potential of cannabinoids.

Adaptive optics is a technique for correcting aberrations and improving image quality. When adaptive optics was first used in microscopy, it was common to rely on iterative approaches to determine the aberrations present. It is advantageous to avoid iteration, and therefore there has been a shift to deep learning for aberration prediction. However, issues remain regarding the practicalities of machine learning for adaptive optics, an important one being the requirement for a large training dataset. Here, we explore transfer learning to overcome this need for data by pre-training a network on a large simulated dataset and fine-tuning it with reduced experimental data for application in an experimental setting. We demonstrate that the pre-trained network can make noticeable improvements with fine-tuning on just 24 experimental samples. To further enhance practicality, we significantly extend the range of aberrations present, predicting up to 25 Zernike modes with each coefficient ranging from −1 to 1, and perform a thorough analysis of the type and magnitude of phase-diversity required in the input data for a successful network. Our approach demonstrates substantial aberration reduction on experimental data for 10 Zernike modes, with an average 73% decrease in RMS wavefront error from 1.81 to 0.48 rad when correction is applied. This method achieves complete experimental image capture and aberration inference at rates comparable to the image acquisition time of a typical laser scanning microscope. Additionally, we consider the benefits of further improvements via an iterative step. As such, this work addresses some of the key practical hurdles that remain in the use of deep learning for aberration prediction and correction.

The extracellular matrix changes dramatically during the progression of diseases like cancer. These complex, tissue-specific changes are not adequately replicated by most current biomaterial disease models. This work demonstrates, for the first time, a biomaterial system allowing combined, independent control over stiffness, extracellular matrix composition and 3D collagen architecture. Defined hydrogel formulations are successfully perfused into ice-templated collagen scaffolds, controlling the composition of these hybrid scaffolds at constant stiffness. The Young's moduli of these hybrid scaffolds can also be tuned independently of composition via chemical cross-linking. Encapsulation of human dermal fibroblasts reveals that fibroblast morphology depends on hybrid scaffold composition and on viscoelasticity, highlighting the importance of a system that decouples biophysical from biochemical properties. Finally, these hybrid scaffolds are successfully applied to exert combined control over biochemical and biophysical drivers of cell growth and invasion, focusing on breast cancer as proof-of-concept. The results reveal that collagen fiber patterning enhances breast cancer cell proliferation, also directing the invasion of patient-derived breast cancer cells. These hybrid scaffolds are therefore promising new tools for dissecting the diverse but complementary roles played by the extracellular matrix in regulating cell phenotype, in a range of healthcare applications.

This study investigated the relationships between emulsion droplet triacylglycerol (TAG) crystallinity and colloidal acid stability on in vitro digestion microstructure, lipolysis, and docosahexaenoic acid (DHA) bioaccessibility. Oil-in-water (o/w) nanoemulsions (20 wt%) composed of 50/50 DHA-rich algal oil with either palm stearin (PS) or olein (PO), and either acid-stable Tween 80 (2.0 wt%; AS) or acid-unstable soy lecithin (2.2 wt%; AU) were fast or slow cooled to 37 °C after microfluidization. Similar particle size distributions and D3,2 (~131–142 nm) and D4,3 (~208–239 nm) values were achieved. All emulsions were highly electronegative (~−45–70 mV) and differences (p < 0.05) were due to emulsifier type, as expected, and cooling rate. Next, emulsions were subjected to INFOGEST in vitro digestion for analysis of intestinal lipolysis by free fatty acid titration and DHA bioaccessibility. As expected, AU emulsions flocculated, forming larger aggregates during the gastric phase. Slower lipolysis was observed for the AU emulsions (p < 0.05), attributed to gastric phase aggregation, and lower 2 h lipolysis was observed for the PS emulsions (~74–77%) based on the presence of crystallinity. DHA bioaccessibility was high (~57–88%), especially for the AS emulsions (p < 0.05). Therefore, emulsion colloidal acid stability and TAG physical state significantly impacted emulsion gastric microstructure, digestion, and bioaccessibility.

Stiffening of tissue is a hallmark of cancer progression, promoting invasive phenotypes through altered cell-extracellular matrix (ECM) interactions. However, how fully formed epithelial structures respond to mechanical cues within their native ECM environment remains poorly understood. Here, using a 3D in situ stiffening hydrogel system that enables modulation of stiffness around mature normal mammary acini, it uncovers critical steps in ECM remodeling and invasion of epithelial structures and discover molecular mechanisms driving this process. Stiffening around mature acini triggers two temporally distinct phases of epithelial remodeling, a rapid priming phase involving basement membrane (laminin, LN) disruption and fibronectin (FN) secretion, followed by a delayed invasion phase characterized by FN remodeling and LN re-deposition that coincides with acinar proliferation and invasion. Mechanistically, it is shown that these changes are mediated by α3β1- and α5β1-integrin-focal adhesion kinase (FAK) signaling, which in turn activates the mechanosensitive ion channel Piezo1 to regulate ECM composition, remodeling, and acinar invasion. Together, the findings reveal how mature epithelial structures dynamically respond to mechanical stiffening to create an invasive niche, offering new insights into how tissue architecture and stiffness synergize to drive breast cancer progression.

In dividing cells, chromosomes are coated in a sheath of proteins and RNA called the mitotic chromosome periphery. This sheath is thought to confer biophysical properties to chromosomes, critical for successful cell division. However, the details of chromosome mechanics, and specifically, if and how the chromosome periphery contributes to them, remain poorly understood. In this study, we present a comprehensive characterisation of single-chromosome mechanics using optical tweezers and an improved broadband microrheology analysis. We extend this analysis to direct measurements of the chromosome periphery by manipulating levels of Ki-67, its chief organiser, and apply a rheological model to isolate its contribution to chromosome mechanics. We report that the chromosome periphery governs dynamic self-reorganisation of chromosomes and acts as a structural constraint, providing force-damping properties. This work provides significant insight into chromosome mechanics and will inform our understanding of the mitotic chromosome periphery's role in cell division.

Microrheology with optical tweezers (MOT) is an all-optical technique that allows the user to investigate a materials’ viscoelastic properties at microscopic scales, and is particularly useful for those materials that feature complex microstructures, such as biological samples. MOT is increasingly being employed alongside 3D imaging systems and particle tracking methods to generate maps showing not only how properties may vary between different points in a sample but also how at a single point the viscoelastic properties may vary with direction. However, due to the diffraction limited shape of focussed beams, optical traps are inherently anisotropic in 3D. This can result in a significant overestimation of the fluids’ viscosity in certain directions. As such, the rheological properties can only be accurately probed along directions parallel or perpendicular to the axis of trap beam propagation. In this work, a new analytical method is demonstrated to overcome this potential artefact. This is achieved by performing principal component analysis on 3D MOT data to characterise the trap, and then identify the frequency range over which trap anisotropy influences the data. This approach is initially applied to simulated data for a Newtonian fluid where the trap anisotropy induced maximum error in viscosity is reduced from ~ 150% to less than 6%. The effectiveness of the method is corroborated by experimental MOT measurements performed with water and gelatine solutions, thus confirming that the microrheology of a fluid can be extracted reliably across a wide frequency range and in any arbitrary direction. This work opens the door to fully spatially and angularly resolved 3D mapping of the rheological properties of soft materials over a broad frequency range.

Machado-Joseph disease (MJD), also known as spinocerebellar ataxia type 3, is a fatal neurodegenerative disease that causes loss of balance and motor co-ordination, eventually leading to paralysis. It is caused by the autosomal dominant inheritance of a long CAG trinucleotide repeat sequence within the ATXN3 gene, encoding for an expanded polyglutamine (polyQ) repeat sequence within the ataxin-3 protein. Ataxin-3 containing an expanded polyQ repeat is known to be highly prone to intraneuronal aggregation, and previous studies have demonstrated that protein quality control pathways, such as autophagy, are impaired in MJD patients and animal models of the disease. In this study, we tested the therapeutic potential of spermidine on zebrafish and rodent models of MJD to determine its capacity to induce autophagy and improve functional output. Spermidine treatment of transgenic MJD zebrafish induced autophagy and resulted in increased distances swum by the MJD zebrafish. Interestingly, treatment of the CMVMJD135 mouse model of MJD with spermidine added to drinking water did not produce any improvement in motor behaviour assays, neurological testing or neuropathology. In fact, wild type mice treated with spermidine were found to have decreased rotarod performance when compared to control animals. Immunoblot analysis of protein lysates extracted from mouse cerebellar tissue found little differences between the groups, except for an increased level of phospho-ULK1 in spermidine treated animals, suggesting that autophagy was indeed induced. As we detected decreased motor performance in wild type mice following treatment with spermidine, we conducted follow up studies into the effects of spermidine treatment in zebrafish. Interestingly, we found that in addition to inducing autophagy, spermidine treatment also induced apoptosis, particularly in wild type zebrafish. These findings suggest that spermidine treatment may not be therapeutically beneficial for the treatment of MJD, and in fact warrants caution due to the potential negative side effects caused by induction of apoptosis.

Significance: Skin color affects light penetration leading to differences in its absorption and scattering properties. COVID-19 highlighted the importance of understanding of the interaction of light with different skin types, e.g., pulse oximetry (PO) unreliably determined oxygen saturation levels in people from Black and ethnic minority backgrounds. Furthermore, with increased use of other medical wearables using light to provide disease information and photodynamic therapies to treat skin cancers, a thorough understanding of the effect skin color has on light is important for reducing healthcare disparities. Aim: The aim of this work is to perform a thorough review on the effect of skin color on optical properties and the implication of variation on optical medical technologies. Approach: Published in vivo optical coefficients associated with different skin colors were collated and their effects on optical penetration depth and transport mean free path (TMFP) assessed. Results: Variation among reported values is significant. We show that absorption coefficients for dark skin are similar to 6%similar to 6% to 74% greater than for light skin in the 400 to 1000 nm spectrum. Beyond 600 nm, the TMFP for light skin is greater than for dark skin. Maximum transmission for all skin types was beyond 940 nm in this spectrum. There are significant losses of light with increasing skin depth; in this spectrum, depending upon Fitzpatrick skin type (FST), on average 14% to 18% of light is lost by a depth of 0.1 mm compared with 90% to 97% of the remaining light being lost by a depth of 1.93 mm. Conclusions: Current published data suggest that at wavelengths beyond 940 nm light transmission is greatest for all FSTs. Data beyond 1000 nm are minimal and further study is required. It is possible that the amount of light transmitted through skin for all skin colors will converge with increasing wavelength enabling optical medical technologies to become independent of skin color.

Medical imaging is advancing rapidly through the development of novel laser sources and non-linear imaging methodologies. These developments are boosting deep tissue imaging allowing researchers to study diseases deep in the body enabling early diagnosis and better treatment. To help with the testing and optimization of these imaging systems and to aid in this process of deep tissue imaging, it's important to have robust, stable and reproducible standards and phantoms. Herein we present the design and fabrication of robust, multi-layered, hydrogel-based standards. The hydrogel used is a double network hydrogel consisting of two interpenetrating networks agarose and polyacrylamide. Thin layers of tough double network hydrogels are stacked to form multilayered depth standards having modality specific signaling markers embedded in between. Standard design and assembly ensured long term stability and easy transport. These proved useful in-depth imaging studies, utilizing multiple imaging modalities, including one photon fluorescence (1PEF), two photon fluorescence (2PEF), coherent anti-Stokes Raman imaging (CARS) and second harmonic generation imaging (SHG).

Mitotic chromosomes are specialised packets of condensed genetic material with dynamic mechanical properties. Each chromosome is coated by a sheath of proteins and RNA, the mitotic chromosome periphery (MCP). The MCP is widely considered as an essential chromosome compartment where its multiple functions bestow material properties important for successful cell division. However, the details of the micromechanical properties of mitotic chromosomes, and specifically if and how the MCP contributes to these features, remain poorly understood. In this study, we present the most comprehensive characterisation of single-chromosome mechanics to date spanning a broadband frequency range, using optical tweezers and a novel microrheology technique. We extend this analysis to the first direct measurements of MCP micromechanics by manipulating levels of Ki-67, the chief organiser of this compartment, and apply a rheological model to isolate its contribution to chromosome dynamics. We report that the MCP governs high-frequency self-reorganisation dynamics and acts as a structural constraint, providing force-damping properties that mitigate mitotic stress. This work significantly advances our understanding of chromosome micromechanics and how the MCP contributes to the fundamental properties of chromosomes. ### Competing Interest Statement The authors have declared no competing interest.

Sprouted wheat wholemeal was reported to enhance the nutritional and sensory properties of cereal products, but few human studies exist. The effect of blending 50 % sprouted wheat wholemeal in a bread recipe on the postprandial glycemic and satiety responses, and sensory-related sensations was investigated in this randomized crossover human study with 12 healthy participants. Capillary blood samples were collected and glycemic response was determined at 0, 15, 30, 45, 60, 90, 120 min. Satiety visual analogue scales were given every 30 min. While substituting bread wheat flour with sprouted wheat wholemeal significantly increased the α-amylase activity in the dough (p < 0.05), it did not alter in vitro digestibility or postprandial glycemic and satiety responses (p > 0.05). Likewise, participant overall acceptability was not adversely affected. Sprouted wheat wholemeal can be used as a functional ingredient in breadmaking, although it did not, in this study, significantly affect digestibility parameters.

Understanding the mechanisms that drive TDP-43 pathology is integral to combating amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD) and other neurodegenerative diseases. Here we generated a longitudinal quantitative proteomic map of the cortex from the cytoplasmic TDP-43 rNLS8 mouse model of ALS and FTLD, and developed a complementary open-access webtool, TDP-map ( https://shiny.rcc.uq.edu.au/TDP-map/ ). We identified distinct protein subsets enriched for diverse biological pathways with temporal alterations in protein abundance, including increases in protein folding factors prior to disease onset. This included increased levels of DnaJ homolog subfamily B member 5, DNAJB5, which also co-localized with TDP-43 pathology in diseased human motor cortex. DNAJB5 over-expression decreased TDP-43 aggregation in cell and cortical neuron cultures, and knockout of Dnajb5 exacerbated motor impairments caused by AAV-mediated cytoplasmic TDP-43 expression in mice. Together, these findings reveal molecular mechanisms at distinct stages of ALS and FTLD progression and suggest that protein folding factors could be protective in neurodegenerative diseases.

A method to extract scattering coefficients from random media is presented. A deep learning network is trained from Monte Carlo simulations. Using angular and spatial information together greatly improved robustness and accuracy over previous approaches. (tel: +44 7536964914, e-mail: eexyz67@nottingham.ac.uk ).

Optical imaging quality can be severely degraded by system and sample induced aberrations. Existing adaptive optics systems typically rely on iterative search algorithm to correct for aberrations and improve images. This study demonstrates the application of convolutional neural networks to characterise the optical aberration by directly predicting the Zernike coefficients from two to three phase-diverse optical images. We evaluated our network on 600,000 simulated Point Spread Function (PSF) datasets randomly generated within the range of -1 to 1 radians using the first 25 Zernike coefficients. The results show that using only three phase-diverse images captured above, below and at the focal plane with an amplitude of 1 achieves a low RMSE of 0.10 radians on the simulated PSF dataset. Furthermore, this approach directly predicts Zernike modes simulated extended 2D samples, while maintaining a comparable RMSE of 0.15 radians. We demonstrate that this approach is effective using only a single prediction step, or can be iterated a small number of times. This simple and straightforward technique provides rapid and accurate method for predicting the aberration correction using three or less phase-diverse images, paving the way for evaluation on real-world dataset.

Biomechanical cues from the extracellular matrix (ECM) are essential for directing many cellular processes, from normal development and repair, to disease progression. To better understand cell-matrix interactions, we have developed a new instrument named 'OptoRheo' that combines light sheet fluorescence microscopy with particle tracking microrheology. OptoRheo lets us image cells in 3D as they proliferate over several days while simultaneously sensing the mechanical properties of the surrounding extracellular and pericellular matrix at a sub-cellular length scale. OptoRheo can be used in two operational modalities (with and without an optical trap) to extend the dynamic range of microrheology measurements. We corroborated this by characterising the ECM surrounding live breast cancer cells in two distinct culture systems, cell clusters in 3D hydrogels and spheroids in suspension culture. This cutting-edge instrument will transform the exploration of drug transport through complex cell culture matrices and optimise the design of the next-generation of disease models.

TAR DNA binding protein 43 (TDP-43) pathology is a key feature of over 95% of amyotrophic lateral sclerosis (ALS) and nearly half of frontotemporal dementia (FTD) cases. The pathogenic mechanisms of TDP-43 dysfunction are poorly understood, however, activation of cell stress pathways may contribute to pathogenesis. We, therefore, sought to identify which cell stress components are critical for driving disease onset and neurodegeneration in ALS and FTD. We studied the rNLS8 transgenic mouse model, which expresses human TDP-43 with a genetically-ablated nuclear localisation sequence within neurons of the brain and spinal cord resulting in cytoplasmic TDP-43 pathology and progressive motor dysfunction. Amongst numerous cell stress-related biological pathways profiled using qPCR arrays, several critical integrated stress response (ISR) effectors, including CCAAT/enhancer-binding homologous protein (Chop/Ddit3) and activating transcription factor 4 (Atf4), were upregulated in the cortex of rNLS8 mice prior to disease onset. This was accompanied by early up-regulation of anti-apoptotic gene Bcl2 and diverse pro-apoptotic genes including BH3-interacting domain death agonist (Bid). However, pro-apoptotic signalling predominated after onset of motor phenotypes. Notably, pro-apoptotic cleaved caspase-3 protein was elevated in the cortex of rNLS8 mice at later disease stages, suggesting that downstream activation of apoptosis drives neurodegeneration following failure of early protective responses. Unexpectedly, suppression of Chop in the brain and spinal cord using antisense oligonucleotide-mediated silencing had no effect on overall TDP-43 pathology or disease phenotypes in rNLS8 mice. Cytoplasmic TDP-43 accumulation therefore causes very early activation of ISR and both anti- and pro-apoptotic signalling that switches to predominant pro-apoptotic activation later in disease. These findings suggest that precise temporal modulation of cell stress and death pathways may be beneficial to protect against neurodegeneration in ALS and FTD.