The majority of drugs are typically orally administered. The journey from drug discovery to approval is often long and expensive, involving multiple stages. A major challenge in the drug development process is drug-induced liver injury (DILI), a condition that affects the liver, the organ responsible for metabolizing most drugs. Traditionally, identifying DILI risk has been difficult due to the poor correlation between preclinical animal models and in vitro systems. Differences in physiology between humans and animals or cell lines contribute to the failure of many drug programs during clinical trials. The use of advanced in vitro systems that closely mimic human physiology, such as organ-on-a-chip models like gut-liver-on-a-chip, can be crucial in improving drug efficacy while minimizing toxicity. Additionally, the adaptation of these technologies has the potential to significantly reduce both the time and cost associated with obtaining safe drug approvals, all while adhering to the 3Rs principle (replacement, reduction, refinement). In this review, we discuss the significance, current status, and future prospects of advanced platforms, specifically organ-on-a-chip models, in supporting preclinical drug discovery.

Metabolic health is tightly regulated by neuro-hormonal control, and systemic metabolic dysfunction may arise from altered function of the hypothalamic-anterior pituitary axis (HAPA). Ancient experimental observations of hypothalamic obesity (HO) and liver cirrhosis occurring among animals subjected to hypothalamic injury can now be explained using the more recent concepts of lipotoxicity and metabolic dysfunction-associated steatotic liver disease (MASLD). Lipotoxicity, the range of abnormalities resulting from the harmful effects of fatty acids accumulated in organs outside of adipose tissue, is the common pathogenic factor underlying closely related conditions like hypothalamic syndrome, HO, and MASLD. The hormonal deficits and the array of metabolic and metabolomic disturbances that occur in cases of HO are discussed, along with the cellular and molecular mechanisms that lead, within the MASLD spectrum, from uncomplicated steatotic liver disease to steatohepatitis and cirrhosis. Emphasis is placed on knowledge gaps and how they can be addressed through novel studies. Future investigations should adopt precision medicine approaches by precisely defining the hormonal imbalances and metabolic dysfunctions involved in each individual patient with HO, thus paving the way for tailored management of MASLD that develops in the context of altered HAPA.

A diallel cross involving 10 I 1 red clover ( Trifolium pratense L.) clones was evaluated for survival on four dates over a 3‐year period, 1973‐75, and vigor of growth on three dates in 1973 and 1974. Significant nonadditive genetic variance was found for survival in 1974 and 1975 and for vigor in 1973. This nonadditive portion of genetic variance was substantially greater than in a previous study involving I 0 clones and strengthens the case for production of hybrid red clover. Significant reciprocal effects were found for all traits except vigor in 1974. GCA effects of I 1 clones failed to agree with GCA effects of their I 0 parents in the previous study, possibly owing to chance selection of inferior I 1‐ segregates from superior I 0 clones, or to genotype ✕ environment interactions.

Mitochondria not only are a source of reactive oxygen species (ROS) but also are sites of oxidative damage. In plants, mitochondria must normally operate when there are high levels of ROS produced during photosynthesis and photorespiration. These levels are further enhanced during biotic and abiotic stress of plants. Excessive stress can lead to mitochondrial damage, which may then lead to induction of programmed cell death in plants. We outline methods for imposing oxidative stress in plants, provide methods for measurements of its severity, and then explain assays for assessing plant mitochondrial oxidative damage and measuring the capacity of key stress defense and response pathways.

Aims: The latent transforming growth factor-β binding proteins (LTBP) are a family of widely expressed multidomain glycoproteins. cDNA for four different LTBP (LTBP–1, –2, –3, –4) was cloned from different sources and also from different species. The LTBP genes set up a subfamily of the extracellular fibrillin proteins, which constitute the backbone of extracellular filaments. We focused our work on LTBP–1 which is expressed in different organs [1], forms intracellular complexes with transforming growth factor-β (TGF-β1, –2, –3), target the growth factor to the extracellular matrix, and is a key player in controlling wound healing. Methods: We generated a mouse model system, in which both isoforms of the LTBP–1 gene, the short (LTBP–1S) and the long isoform (LTBP–1L), were deleted. We evaluated in detail liver sections from wild type and knock out littermates by Sirius Red staining as well as the measurement of serum parameters (AST, ALT, bilirubin) relevant for liver function. In addition, RT-PCR analysis was performed to monitor the expression level of the other LTBP isoforms (LTBP–2, –3, –4) in cultured hepatic stellate cells (HSC) and skin fibroblasts. Comparative gene expression profiles from HSC were generated using PIQOR™ Immunology Microarrays (Miltenyi Biotec). Results: Delicate differences between wild type and knockout animals were detectable in bone morphology. Generally, the head was more compact in LTBP–1 deficient mice than in normal controls. In cultured HSC and skin fibroblasts, the relative transcript levels of other LTBP isoforms were not altered as proven by RT-PCR. Microarray analysis substantiated the crucial influence of LTBP–1 on TGF-β-dependent genes and lead to the identification of novel gene targets controlled by the LTBP–1/TGF-β network. Conclusions: The staining of liver tissues from LTBP–1 nulls displayed, that the absence of LTBP–1 does not induce any signs of hepatic dysfunction. However, our microarray data analysis clearly demonstrates that LTBP–1-deficiency is strongly associated with differences in expressing and controlling of various TGF-β-dependent genes. Therefore, we conclude that the LTBP–1 deficiency will be critical in situations when TGF-β signalling is pronounced, i.e. fibrogenesis.

The FAIR (findable, accessible, interoperable, and re-usable) principles and practice recommendations provide high level guidance and recommendations that are not research-domain specific in nature. There remains a gap in practice at the data provider and domain scientist level demonstrating how the FAIR principles can be applied beyond a set of generalist guidelines to meet the needs of a specific domain community. We present our insights developing FAIR thresholds in a domain specific context for self-governance by a community (agricultural research). ‘Minimum thresholds’ for FAIR data are required to align expectations for data delivered from providers’ distributed data stores through a community-governed federation (the Agricultural Research Federation, AgReFed). Data providers were supported to make data holdings more FAIR. There was a range of different FAIR starting points, organisational goals, and end user needs, solutions, and capabilities. This informed the distilling of a set of FAIR criteria ranging from ‘Minimum thresholds’ to ‘Stretch targets’. These were operationalised through consensus into a framework for governance and implementation by the agricultural research domain community. Improving the FAIR maturity of data took resourcing and incentive to do so, highlighting the challenge for data federations to generate value whilst reducing costs of participation. Our experience showed a role for supporting collective advocacy, relationship brokering, tailored support, and low-bar tooling access particularly across the areas of data structure, access and semantics that were challenging to domain researchers. Active democratic participation supported by a governance framework like AgReFed’s will ensure participants have a say in how federations can deliver individual and collective benefits for members.

The global need for increased food production means that agriculture is moving into regions with lower rainfall and saline soils, which occupy over 6% of the world land area (Munns, 2005). The aim of this workshop was to work towards completing an energy budget for the mechanisms of salinity tolerance in crop plants, as a guide to the most cost-effective breeding strategies for increasing salt tolerance and yield of important crops. The discussions took into account the supply of energy from mitochondria and chloroplasts, energy demands for transport of water, Na+, Cl− and K+, as well as for processes involved in growth and osmotic adjustment. The premise of the workshop was that by undertaking an energy budget of salinity tolerance using our current understanding of transport processes and measurements of fluxes, including respiratory fluxes through the cytochrome vs alternative respiratory pathways, we would test our understanding and/or reveal deficiencies in the measurements (Fig. 1). Such energy budget considerations have indicated how energy savings are achieved in rice coleoptiles under the combined stresses of anoxia and salinity (Kurniasih et al., 2017). Understanding energy costs/benefits of specific components will also provide a better foundation for engineering salt tolerance; for example, is it better to increase the number of transporters expressed or the energy efficiency of transport (Greenway & Munns, 1983)? It is becoming increasingly evident that a detailed knowledge of the role, location and mechanism of proteins that help confer salt tolerance is needed. For example, the rice Na+ transporter OsHKT1;4, which prevents Na+ accumulating in shoots to toxic levels, when constitutively overexpressed reduces leaf Na+ but results in higher root Na+ and reduced salt tolerance (Oda et al., 2018). Two major paradoxes that underlie the need to examine the energetics of salinity tolerance were identified. (1) Within nonhalophyte species, genetic variation in salt tolerance is often associated with lower leaf Na+ concentration, that is, with Na+ exclusion from leaves. This presents a paradox in terms of energy use efficiency because osmotic adjustment using Na+ would seem a cheaper option than using organic solutes, for example c. 30 mol ATP mol−1 hexose is locked up (Munns & Gilliham, 2015). This implies that there are significant costs in nonhalophytes of having high Na+ concentrations in leaves. The costs are presumably for transport and intracellular compartmentation. A poor capacity for vacuolar compartmentation (Bonales-Alatorre et al., 2013) or chloroplast exclusion (Bose et al., 2017) could also be a factor in many nonhalophytes. (2) Previous calculations of the energy cost for root membrane transport of Na+, as well as nutrients required for growth such as NO3−, appear to exceed the energy that is available according to our current understanding of the transport processes (Kurimoto et al., 2004; Malagoli et al., 2008). The way that energy budgets can be determined and the components that were discussed are indicated in Fig. 2. Major conclusions from the workshop were: In conclusion, it was agreed that biophysical modelling of salt and water transport in cells (Foster & Miklavcic, 2015), roots (Foster & Miklavcic, 2017) and leaves, incorporating the known transporters and ion gradients, would greatly facilitate our understanding of the energy costs of salinity tolerance in crop plants. These models would also inform experimentalists of the types of measurements required and standardization of units and the normalization of fluxes. The participants acknowledge the financial assistance from the Australian Research Council Centre of Excellence in Plant Energy Biology (CE140100008), the Australian Research Council Industrial Transformation Hub Legumes for Sustainable Agriculture (IH140100013), and the logistical assistance of Rebecca Vandeleur.

1. The Isolation of Plant Organelles and Structures in the Post-Genomics Era A. Harvey Millar and Nicolas L. Taylor 2. Approaches to Characterize Organelle, Compartment, or Structure Purity Stefanie J. Mueller, Sebastian N.W. Hoernstein, and Ralf Reski Part I: Isolation of Organelles and Organelle Compartments < 3. Isolation of Nuclei and Nucleoli Alison F. Pendle and Peter J. Shaw 4. Isolation and Suborganellar Fractionation of Arabidopsis Chloroplasts Ursula Flores-Perez and Paul Jarvis 5. Isolation of Chromoplasts and Sub-Organellar Compartments from Tomato and Bell Pepper Fruit Cristina Barsan, Marcel Kuntz, and Jean-Claude Pech 6. Leucoplast Isolation and Subfractionation William C. Plaxton 7. Isolation of Mitochondria, Their Sub-Organellar Compartments, and Membranes Owen Duncan, A. Harvey Millar, and Nicolas L. Taylor 8. Isolation of Arabidopsis Leaf Peroxisomes and the Peroxisomal Membrane Sigrun Reumann and Piotr Lisik 9. Isolation of Vacuoles and the Tonoplast Jan Zouhar 10. Isolation of Endoplasmic Reticulum and Its Membrane G. Eric Schaller 11. Enrichment of Golgi Membranes from Triticum aestivum (Wheat) Seedlings Wei Zeng, Berit Ebert, Harriet T. Parsons, Carsten Rautengarten, Antony Bacic, and Joshua L. Heazlewood 12. Isolation of Autolysosomes from Tobacco BY-2 Cells Chihiro Takatsuka, Yuko Inoue, and Yuji Moriyasu 13. Isolation of Protein Storage Vacuoles and Their Membranes Tomoo Shimada and Ikuko Hara-Nishimura Part II: Isolation of Other Structures and Compartments < 14. Isolation of the Cell Wall Herve Canut, Cecile Albenne, and Elisabeth Jamet 15. Isolation of Plasmodesmata Christine Faulkner and Emmanuelle M.F. Bayer 16. Isolation of Plasma Membrane and Plasma Membrane Microdomains Anzu Minami, Daisuke Takahashi, Yukio Kawamura, and Matsuo Uemura 17. Enrichment of the Plant Cytosolic Fraction Jeemeng Lao, Andreia M. Smith-Moritz, Jennifer C. Mortimer, and Joshua L. Heazlewood 18. Isolation of Apoplast Erik Andreasson, Kibrom B. Abreha, and Svante Resjoe 19. Isolation of Cytosolic Ribosomes Hanna Klang Arstrand and Maria V. Turkina 20. Isolation of Plastid Ribosomes Kenichi Yamaguchi 21. Isolation of Mitochondrial Ribosomes Adam J. Carroll 22. Isolation of Microtubules and Microtubule-Associated Proteins Takahiro Hamada and Seiji Sonobe 23. Isolation of Actin and Actin Binding Proteins Etsuo Yokota 24. Purification of 26S Proteasomes and Their Sub-Complexes from Plants Richard S. Marshall, David C. Gemperline, and Richard D. Vierstra