The influence of buttermilk or buttermilk powder addition to cheese milk or cheese curds respectively on cheese functional properties, free fatty acid profiles and subsequent volatile and sensory characteristics was investigated. Buttermilk addition to cheese milk resulted in a softer cheese compared to other cheeses, with a significantly reduced flowability, while buttermilk powder addition had no influence on cheese firmness but cheese flowability was also reduced compared to the control cheese. Larger pools of free fat, higher levels of free fatty acids, volatile compounds and significant differences in sensory profiles associated with off-flavour were also observed with the addition of buttermilk to cheese milk. Application of light microscopy, using toluidine blue stain, facilitated the visualisation of fat globule structure and distribution within the protein matrix. Addition of 10% buttermilk powder resulted in significant increases in volatile compounds originating from proteolysis pathways associated with roasted, green aromas. Descriptive sensory evaluation indicated few differences between the 10% buttermilk powder and the control cheese, while buttermilk cheeses scored negatively for sweaty, barnyard aromas, oxidized and off flavors, correlating with associated volatile aromas. Addition of 10% buttermilk powder to cheese curds results in cheese comparable to the control Cheddar with some variations in volatile compounds resulting in a cheese with similar structural and sensory characteristics albeit with subtle differences in overall cheese flavor. This could be manipulated to produce cheeses of desirable quality, with potential health benefits due to increased phospholipid levels in cheese.

Diabetic Kidney Disease (DKD) represents a significant global health burden and is recognised as the leading cause of end-stage renal disease. Kidney organoids derived from human induced Pluripotent Stem Cells (hiPSCs) have the potential to transform how we model renal disease and may provide personalised replacement tissues for patients with renal failure. However, kidney organoids remain poorly reproducible, and are structurally and functionally immature. Three-dimensional cultures that more appropriately mimic the complexity of the in vivo microenvironment are required to improve organoid maturation and structural authenticity. Here, we describe the application of semi-synthetic Gelatin Methacryloyl (GelMA) hydrogels as extracellular support matrices for the differentiation of hiPSC-derived kidney organoids. Hydrogels of defined mechanical strengths were generated by varying the concentration of GelMA solution in the presence of low concentration photo-initiator. After confirming a high level of mechanical stability of the hydrogels over extended culture periods, their effect on kidney organoid maturation was investigated. Organoids differentiated within GelMA hydrogels generated typical renal cell types including podocytes, tubular epithelia, renal interstitial cells, and some nascent vascularisation. Interestingly, kidney organoids derived within hydrogels that closely approximate the stiffness of the adult human kidney (∼5000-10,000 Pa) demonstrated improved podocyte maturation and were shown to upregulate renal vesicle-associated genes at an earlier stage following encapsulation when compared to organoids derived within softer hydrogels (∼400 Pa). A model of TGFβ-induced injury was also developed to investigate the influence of the mechanical environment in propagating early, fibrotic-like features of DKD within organoids. Growth within the softer matrix was shown to reduce pSMAD3 expression following TGFβ1 treatment, and accordingly ameliorate the expression of the myofibroblast marker α-Smooth Muscle Actin (α-SMA). This work demonstrates the suitability of GelMA hydrogels as mechanically-stable, highly-tuneable, batch-to-batch reproducible three-dimensional supports for hiPSC-derived kidney organoid growth and differentiation, and further substantiates the role of the biophysical environment in guiding processes of cell fate determination and organoid maturation.

TGFβ1 plays a regulatory role in the determination of renal cell fate and the progression of renal fibrosis. Here we show an association between SMAD3 and the histone methyltransferase, EZH2, during cell differentiation; ChIP-seq revealed that SMAD3 and EZH2 co-occupy the genome in iPSCs and in iPSC-derived nephron progenitors. Through integration of single cell gene expression and epigenome profiling, we identified de novo ACTA2^+ve/POSTN^+vemyofibroblasts in kidney organoids treated with TGFβ1, characterised by increased SMAD3-dependentcischromatin accessibility and gene expression associated with fibroblast activation. We have identified fibrosis-associated regulons characterised by enrichment of SMAD3, AP1, the ETS family of transcription factors, and NUAK1, CREB3L1, and RARG, corresponding to enriched motifs at accessible loci identified by scATACseq. Treatment with the EZH2 specific inhibitor GSK343, blocked SMAD3-dependentcisco-accessibility and inhibited myofibroblast activation. This mechanism, through which TGFβ signals directly to chromatin, represents a critical determinant of fibrotic, differentiated states.

Background: Potassium bromate (KBrO3), a food additive, has been used in many bakery products as an oxidizing agent. It has been shown to induce renal cancer in many in-vitro and in-vivo experimental models. Objectives: This study evaluated the carcinogenic potential of potassium bromate (KBrO3) and the chemopreventive mechanisms of the anti-oxidant and anti-inflammatory phytochemical, curcumin against KBrO3-induced carcinogenicity. Method: Lactate dehydrogenase (LDH) cytotoxicity assay and morphological characteristics were used to assess curcumin's cytoprotective potential against KBrO3 toxicity. To assess the chemopreventive potential of curcumin against KBrO3-induced oxidative insult, intracellular H2O2 and the nuclear concentration of the DNA adduct 8- OHdG were measured. PCR array, qRT-PCR, and western blot analysis were used to identify dysregulated genes by KBrO3 exposure. Furthermore, immunofluorescence was used to evaluate the ciliary loss and the disturbance of cellular tight junction induced by KBrO3. Results: Oxidative stress assays showed that KBrO3 increased the levels of intracellular H2O2 and the DNA adduct 8-OHdG. Combination of curcumin with KBrO3 efficiently reduced the level of H2O2 and 8-OHdG while upregulating the expression of catalase. PCR array, qRT-PCR, and western blot analysis revealed that KBrO3 dysregulated multiple genes involved in inflammation, proliferation, and apoptosis, namely CTGF, IL-1, and TRAF3. Moreover, qRT-PCR and immunofluorescence studies showed that KBrO3 negatively affected the tight junctional protein (ZO-1) and induced a degeneration of primary ciliary proteins. The negative impact of KBrO3 on cilia was markedly repressed by curcumin. Conclusion: Curcumin could potentially be used as a protective agent against carcinogenicity of KBrO3.

Prenatal exposure to cocaine causes abnormalities in foetal brain development, which are linked to later development of anxiety, depression and cognitive dysfunction. Previous studies in rodent models have indicated that prenatal cocaine exposure affects proliferation, differentiation and connectivity of neural cell types. Here, using cerebral organoids derived from the human iPSC cell line HPSI1213i-babk_2, we investigated cocaine-induced changes of the gene expression regulatory landscape at an early developmental time point, leveraging recent advances in single cell RNA-seq and single cell ATAC-seq. iPSC-cerebral organoids replicated well-established cocaine responses observed in vivo and provided additional information about the cell-type specific regulation of gene expression following cocaine exposure. Cocaine altered gene expression patterns, in part through epigenetic landscape remodelling, and revealed disordered neural plasticity mechanisms in the cerebral organoids. Perturbed neurodevelopmental cellular signalling and an inflammatory-like activation of astrocyte populations were also evident following cocaine exposure. The combination of altered neuroplasticity, neurodevelopment and neuroinflammatory signalling suggests cocaine exposure can mediate substantial disruption of normal development and maturation of the brain. These findings offer new insights into the cellular mechanism underlying the adverse effects of cocaine exposure on neurodevelopment and point to the possible pathomechanisms of later neuropsychiatric disturbances.

The influence of canal operations on the available habitat and fish communities in conjoined Great Plains streams is not well understood. Three-pass backpack electrofishing was used twice within a three-week period in fall 2018 to identify changes in Turkey Creek (near Kearney, NE) before and after the Kearney Canal was dewatered. Diversity was greater while the canal was operational (t = 398.14, p < 0.001), but community composition remained similar following canal dewatering (J = 62.9%). Species-specific differences between the two time periods were most often observed among cyprinids and centrarchids, and nonnative species were more abundant (z = 6.0, p < 0.001) and comprised a greater percentage of the available biomass (z = 75.7, p < 0.001) when the canal was operational. The wetted width (t = 2.27, p = 0.04) and depth of Turkey Creek (t = 1.90, p = 0.06) significantly decreased after the canal ceased operation. Findings from this case study can be used to further the understanding of fish community responses within streams conjoined with canals and may provide regional water and fish managers important information to improve future management of these systems to support native fishes while simultaneously meeting other water uses for agriculture and recreation.