The Journal of Nutritional Biochemistry

BackgroundDespite the advancements in infant nutrition, a gap still exists in the nutritional composition bioactive ingredients between infant formula and human milk. We developed a next-generation, proof-of-concept infant formula that contains recombinant human milk proteins. ObjectiveTo determine the impact of a novel infant formula (H1) on organ growth and development, and intestinal function compared to donor human milk (DHM) and standard infant formula (S) in a term piglet model. MethodsTerm piglets delivered via cesarean section were fed either a donor human milk (DHM) control, the investigational formula (H1), or infant formula (S) for 10 days. On d 10, a blood sample and tissues were collected. ResultsThere was no difference (P > 0.05) in piglet growth, although H1 piglets had a smaller relative stomach and liver than DHM and S piglets. H1 piglets had higher (P < 0.05) interleukins in the distal ileum, but no other systemic cytokines were elevated compared to the DHM and S piglets. H1 piglet small intestinal histology was similar (P > 0.05) to that of DHM and S piglets. Additionally, H1 piglets had either the same (P > 0.05) or higher (P < 0.05) amino acids in circulation compared to DHM and S piglets. Recombinant human proteins had either similar (P > 0.05) or lower (P < 0.05) activity compared to the native human proteins when assessing the individual ingredients in the H1 formula. ConclusionH1 formula was noninferior to DHM and S based on growth, small intestinal histology and plasma amino acid endpoints when fed to neonatal piglets. These findings warrant further studies to use the neonatal piglet as a model to evaluate more in-depth outcomes of health and safety for new infant formulas. Lay SummaryA novel piglet study shows a hypoallergenic, next-generation infant formula containing recombinant human milk proteins rivals donor human milk and standard formula for growth, gut health, and nutrient status.

Animals experience stressors in a variety of contexts that result in activation of neuroendocrine and cellular stress responses. Release of stress hormones can disrupt or restore redox homeostasis, and the resulting changes in oxidative states, physiology and behavior vary by an individuals stress coping style. However, oxidative stress can also directly modulate neuroendocrine stress signaling. To what extent individual differences in brain antioxidant levels alter behavioral stress levels is not well understood. The present study investigated how N-acetylcysteine amide (NACA), an antioxidant and glutamate-modulating compound, regulates stress behavior across zebrafish (Danio rerio) with different stress coping styles (proactive, reactive). Following 24-hour exposure to NACA or control conditions, we quantified individual and composite stress behaviors using a Light-Dark Test (LDT). As expected, both proactive fish and NACA-treated fish showed significantly lower stress behaviors compared to reactive and control animals, respectively. Notably, stress-reducing effects of NACA were only seen in those with a reactive stress coping style. Overall, our data suggest that antioxidant mechanisms (e.g., glutathione system) may be key in facilitating the distinct behavioral and physiological responses to stressors that characterize alternative stress coping styles. The results underscore how individual differences in stress coping style and redox state can influence behavioral responses to stress.

Environmental enteropathy (EE) is a chronic, subclinical disorder of the small intestine common in low- and middle-income countries (LMICs), where access to sanitation and exposure to enteric pathogens vary greatly by socioeconomic status (SES). Systemic immune cell activation by enteric microbial exposure is a suspected but poorly characterized driver of EE severity. We hypothesised that adults from Low-SES communities would have more severe EE than adults from High-SES communities and that this would be associated with distinct circulating immune cell phenotypes. We enrolled clinically healthy adults from High- (n=26) and Low-SES (n=76) communities in Lusaka, Zambia. Duodenal biopsies from these adults were used for microscopic morphometry assessments, while plasma and stool biomarkers of epithelial damage, intestinal inflammation, microbial translocation, and systemic inflammation were measured by ELISA. Circulating monocyte, neutrophil and T cell phenotypes were characterised in buffy coat cells by flow cytometry. Compared with the High-SES group, adults from Low-SES communities had higher duodenal villus width and crypt depth and lower epithelial surface area, indicative of more severe EE pathology, and higher levels of plasma biomarkers associated with microbial translocation and systemic inflammation. The Low-SES group also had higher expression of activation markers (CD86 and TLR4) and lower expression of HLA-DR on circulating classical monocytes and neutrophils, higher percentages of gut-homing (4{beta}7+) and activated/exhausted (PD-1+) T cells, including gut-homing (4{beta}7+) regulatory T cells. Principal Component Analysis identified key patterns of immune cell phenotypes across SES groups. Confounder-adjusted linear regression models showed that Principal Component 1 (monocyte/neutrophil activation) was inversely associated with duodenal villus height and epithelial surface area across SES groups. These findings indicate that EE severity varies by SES within LMIC and suggest that monocyte and neutrophil activation is linked to greater duodenal remodelling in adults with EE.

Early-life microbiota represent an indispensable factor for the proper development and function of host metabolism and the immune system. We have demonstrated that neonatal exposure to antibiotics for the first 3 weeks (NeoATB) leads to obesity in adulthood, characterized by gut microbiota dysbiosis and dysregulated immune responses. Here, we demonstrate that feeding D-mannose suppresses NeoATB-induced obesity, accompanied by improved glucose tolerance and decreased insulin resistance. Mechanistically, D-mannose feeding decreased hypoxia and increased oxygenation and recovery of metabolic activity of adipocytes. D-mannose restored CD4+Foxp3+ST2+ Tregs, leading to a reduction of Th1 pro-inflammatory cells in the adipose tissue of NeoATB mice. Significantly, we revealed that D-mannose treatment reversed the dysregulated ratios of phylum Firmicutes to phylum Bacteroidetes in obese NeoATB mice, which was surprisingly attributed to D-mannose-mediated suppression of the growth of Firmicutes rather than an increase in the growth of Bacteroidetes. These findings should have therapeutic implications for the treatment of obesity in human patients.

Hemochromatosis and hemosiderosis are iron overload disorders that cause immune dysfunction and increase susceptibility to bacterial infections. There have been numerous case studies reporting septic-like outcomes for hemochromatosis patients infected with enteric Yersiniae; however, research regarding hemosiderosis and Yersinia infection is limited. Here, we have established a mouse model of hemosiderosis by feeding C57BL/6 mice a high-iron diet. These mice exhibit several indicators of iron overload that are seen clinically, including elevated serum iron levels and iron deposition in various tissues. Characterization of the iron overload mouse model shows that a high-iron diet induces local inflammation in the small intestine and systemic inflammation in a time-dependent manner. Oral infection with Yersinia enterocolitica causes complete mortality in the iron-overloaded mice, while wild-type mice all survive and effectively clear the infection. Lastly, we have observed that iron chelation therapies such as Deferoxamine and Deferisarox are detrimental to iron-overloaded mice during Yersinia infection. This work provides a model to further study iron overload disorders and Yersinia infection.

Background and AimsPregnancy requires dynamic, stage-specific adaptations in maternal liver metabolism and growth to sustain fetal development while preserving systemic homeostasis. Estrogen signaling, which significantly increases during pregnancy, is primarily mediated in hepatocytes by estrogen receptor (ER). Although hepatic ER regulates female liver metabolism under non-pregnant conditions, its role in pregnancy-induced hepatic remodeling remains unclear. MethodsWe studied non-pregnant and pregnant control and liver-specific ER knockout (LERKO) mice across gestational stages using longitudinal physiological measurements, liver transcriptomics, targeted metabolomics, histological assessment of cell proliferation, and metabolic phenotyping. ResultsIn control mice, pregnancy elicited sequential hepatic remodeling characterized by early induction of cell-cycle programs, a mid-gestational peak in hepatocyte proliferation with transient suppression of selected metabolic pathways, and late reactivation of specific metabolic programs. Chronic hepatic ER deficiency alters this temporal pattern. LERKO livers showed premature activation of proliferative and anabolic transcriptional programs, changes in amino acid- and fatty acid-related metabolic pathways, and altered temporal regulation of AKT-mTORC1-related signaling. At mid-gestation, LERKO mice displayed reduced hepatocyte proliferation, altered expression of metabolic and insulin-related genes, blunted gestational glucose adaptation without overt evidence of systemic insulin resistance, and changes in the light/dark-phase metabolic patterns. ConclusionsThese findings suggest that hepatic ER is required for the appropriate stage-specific coupling of liver growth, metabolic remodeling, and insulin-responsive signaling during pregnancy. Its loss is associated with gestational hepatic maladaptation and systemic metabolic phenotypes, providing a framework for investigating estrogen-dependent mechanisms underlying pregnancy-associated metabolic and liver disorders. HighlightsHepatic ER is required for stage-specific liver remodeling during pregnancy. Loss of hepatic ER alters temporal coupling of liver growth and metabolism. LERKO mice show early changes in amino acid- and fatty acid-related pathways. Hepatic ER loss reduces proliferation and alters gestational glucose adaptation. Hepatic ER loss is associated with altered light/dark-phase metabolic organization. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/743939v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@d52bborg.highwire.dtl.DTLVardef@b27511org.highwire.dtl.DTLVardef@23b286org.highwire.dtl.DTLVardef@19d9314_HPS_FORMAT_FIGEXP M_FIG C_FIG

Sickness is an organismal response to inflammation, yet its immune, metabolic, neural, and behavioral components are often studied separately and predominantly in male C57BL/6 mice. In this study, we characterized these responses to systemic lipopolysaccharide (LPS) in female BALB/c mice. Mice received intraperitoneal LPS at moderate concentrations and outcomes were assessed during the acute and resolving phases of endotoxemia. LPS caused rapid disappearance of resident peritoneal macrophages, followed by neutrophil accumulation and increased circulating TNF- and IL-6. In the liver, LPS induced inflammatory, acute-phase, and anti-inflammatory transcripts while suppressing genes involved in lipid, cholesterol, and xenobiotic metabolism. Hepatic glutathione was reduced, whereas total superoxide dismutase activity was unchanged. These peripheral responses were followed by transient hypothermia, reduced food intake, and body weight loss. Regional brain mapping showed increased c-Fos labeling in the area postrema, nucleus of the solitary tract, external lateral parabrachial nucleus, paraventricular nucleus of the hypothalamus, and arcuate nucleus. In parallel, LPS selectively promoted IBA1-positive area in the median eminence and arcuate nucleus, whereas several other regions showed no changes, indicating that neuronal and microglial responses are regionally distinct. Behaviorally, LPS reduced locomotion and exploration, increased freezing, and increased forced-swim immobility. Changes in spatial exploration were most pronounced during the acute phase, whereas locomotor suppression and passive stress-coping persisted longer and varied in magnitude with the timing of inflammatory challenge. Together, these findings show that systemic LPS produces a coordinated sickness state in female BALB/c mice that links peripheral inflammation and hepatic metabolic and redox changes with region-specific neuronal and microglial responses, altered thermoregulation and feeding, and behavioral suppression.

Low protein (LP) diets improve metabolic health in rodents and humans. In rodents, LP diets are typically implemented by replacing protein with carbohydrates like sucrose or cornstarch, keeping diets isocaloric. However, humans can choose from many different types of carbohydrate, and how dietary carbohydrate quality - the precise composition of the dietary sugars - impacts the response to dietary protein remains largely unexplored. Here, mice were fed control (21% protein) or LP (7% protein) diets with four different carbohydrate sources: sucrose, a 1:1 glucose/fructose mixture, glucose, or fructose. While LP diets improved metabolic health across all groups in male mice, carbohydrate quality also significantly altered specific health outcomes, with fructose-fed mice having the lowest body weight and adiposity of all control diets. In female mice, responses to LP diets were influenced by carbohydrate quality, with certain sugars inducing a stronger metabolic response to LP diets than previously seen. Finally, in female APP/PS1 mice, a model of Alzheimer's disease, we find that although LP diets reduce A-beta; plaque burden irrespective of carbohydrate type, dietary sugar type does influence spatial memory. Together, these results demonstrate that while dietary protein is a critical determinant of metabolic and neurological health, carbohydrate quality influences these outcomes in a sex-specific manner.

Children of undernourished women have impaired pre- and postnatal growth. Undernourished women and children have a high incidence of environmental enteric dysfunction (EED), an enteropathy characterized by gut barrier dysfunction and systemic inflammation. Here, we employ gnotobiotic mice to compare the effects of bacterial consortia cultured from the duodenal microbiota of Bangladeshi women with EED and their healthy counterparts. Female mice harboring the EED-derived consortium exhibited fetal and placental growth restriction. Transcriptomic and proteomic analyses disclosed pronounced effects of the EED-derived consortium on the decidual component of the maternal-fetal interface involving tissue-resident uterine natural killer (uNK) cells and disruption of TGF-{beta} signaling between uNK and decidual stromal cells. Co-housing mice with EED and healthy consortia ameliorated these effects, disclosing bacterial targets to improve prenatal development.

High-protein diets increase hepatic sulfur amino acid metabolism, but the underlying transcriptional mechanisms remain unclear. This study investigated whether Kruppel-like factor 15 (KLF15) directly regulates cystathionine {gamma}-lyase (CTH), a key enzyme linking methionine transsulfuration to hydrogen sulfide (H2S) and taurine production. Promoter-reporter assays, electrophoretic mobility shift assays, and chromatin immunoprecipitation identified two functional KLF15-binding elements, designated 1-1 and 2-2, within the proximal Cth promoter. Mutation of either element attenuated KLF15-dependent promoter activation, whereas mutation of both largely abolished it. In vivo luciferase imaging further demonstrated that these elements were required for the hepatic transcriptional response to a high-protein diet. KLF15 loss of function reduced high-protein-diet-induced Cth expression and altered the hepatic sulfur amino acid profile. Methionine, cystathionine, and cystine accumulated, whereas taurine production and the high-protein-diet-induced increase in hepatic H2S were attenuated. Gene expression analyses further indicated that KLF15 selectively regulates components of methionine, taurine, and H2S metabolism rather than controlling the entire sulfur metabolic program. Collectively, these findings establish the high-protein diet-KLF15-CTH axis as a physiologically relevant transcriptional pathway that amplifies hepatic sulfur amino acid disposal and directs sulfur toward H2S and taurine production.

Enteric infections caused by Salmonella enterica remain a major global health concern and are increasingly associated with antimicrobial resistance. Therefore, new strategies to combat this important pathogen are needed. The interactions between S. enterica and the human host have been the subject of intense investigation over the last several decades, yet new findings continue to emerge. We previously showed that 15-deoxy-{Delta}12,14-prostaglandin J2 (15d-PGJ2) reduces Salmonella colonization of macrophages, but the mechanisms underlying this protective effect were still unknown. Here, we demonstrate that 15d-PGJ2 limits Salmonella infection by suppressing TLR4 signaling and inflammasome activation. Treatment with 15d-PGJ2 reduced TLR4 expression, NF-{kappa}B activation, iNOS, COX-2, nitric oxide production, IL-1{beta} release, and inflammasome-related targets, including NLRP3 and caspase-1 activity, while only partially reversing macrophage polarization. Combined treatment with the TLR4 antagonist TAK-242 further reduced bacterial colonization of and IL-1{beta} release by macrophages, supporting the involvement of TLR4 signaling in the effects of 15d-PGJ2. During mouse infections, 15d-PGJ2 reduced bacterial burdens in a tissue-dependent manner. Together, these findings demonstrate that 15d-PGJ2 limits Salmonella infection through selective modulation of TLR4 signaling and inflammasome activation.

Inflammatory bowel disease (IBD) is associated with chronic intestinal inflammation and gut microbial dysbiosis, yet effective microbiome-targeted therapeutics remain limited. Here, we investigated the anti-inflammatory and microbiome-modulating activities of metabolites isolated from Garcinia brassii, an endemic species of the Australian Wet Tropics. Five compounds, including a new natural product named garcitine, were isolated and structurally characterised. In human immune cells, garcinol and garcinia biflavonoid 1 significantly suppressed lipopolysaccharide-induced production of IL-1{beta}, IL-6, and TNF without detectable cytotoxicity, while parvifoliol F selectively inhibited IL-1{beta} release. Therapeutic efficacy was further evaluated in a TNBS-induced murine colitis model, where garcinia biflavonoid 1 and parvifoliol F significantly reduced colonic inflammation and improved histopathological outcomes. 16S rRNA sequencing demonstrated that both compounds restored gut microbial homeostasis by reversing colitis-associated dysbiosis and reducing inflammation-associated microbial signatures. Functional pathway prediction further suggested suppression of pro-inflammatory microbial metabolic pathways following treatment. Together, these findings demonstrate that Garcinia-derived metabolites alleviate experimental colitis through coordinated immunomodulatory and microbiome-reprogramming mechanisms and identify garcinia biflavonoid 1 and parvifoliol F as promising candidates for microbiome-targeted IBD therapeutics.

AimThe present study investigated the significance of iron in regulating megakaryopoiesis, by a diet-based intervention in an in-vivo model. MethodsMale C57BL/6 mice, aged 4-5 weeks were fed on varying iron diets. Following sacrifice, blood samples collected in EDTA tubes were used to analyse haematological parameters, and iron content of liver and spleen was assessed by biochemical analyses. Megakaryocyte-erythroid progenitors (MEPs) were isolated from bone marrow by magnetic bead-based selection. RNA isolated from bone marrow cells and MEPs were used for gene expression analyses, and RNA Sequencing to identify differentially expressed genes (DEGs) and associated pathways. ResultsMice fed on an iron-deficient diet had reduced hepatic iron content after 5 weeks (p < 0.01), while both the hepatic and spleen iron content increased after 3 weeks in mice on an iron-rich diet (p < 0.05) and developed iron overloading. Hb and RBC counts increased (p < 0.05) in iron-rich mice and decreased in iron-deficient mice (p < 0.05), which also showed elevated platelet counts (p < 0.01). This may be explained by increased expression of Gata1, Tal1 (p < 0.01) Mds1 and Pdpk1 (p < 0.05) in bone marrow cells from iron-deficient mice. MEPs isolated from these mice showed elevated expression of genes associated with megakaryocytic differentiation, platelet functions, and genes encoding TGF-{beta}R1 and Smad 2,3 and 4. ConclusionsIron deficiency may activate TGF-{beta} signalling and downstream Smad-mediated transcriptional programs within MEPs. This may promote a shift in lineage commitment towards megakaryopoiesis through elevated expression of megakaryopoiesis related genes.

Environmental exposure to endocrine disrupting chemicals, like bisphenol-A (BPA), can impart detrimental effects on developing feto-placental unit, during pregnancy. Placenta remains a central player maintaining this feto-placental homeostasis for sustenance of a healthy pregnancy. Thus, the bisphenol-A mediated endocrine disruption affects the healthy functioning of placenta by altering key processes, such as tissue remodelling, angiogenesis, and metabolism. However, the underlying mechanism of BPA-altered ECM remodelling remains elusive. Therefore, in this study we investigated the BPA mediated changes in placental tissue remodelling using a bisphenol-A exposed murine model during pregnancy. The results reveal that, the phenotypic changes in feto-placental interface correlates with perturbed placental proteome in response to BPA. Further investigation highlights a S100a10-Annexin A2 axis mediated upregulation of tissue plasminogen activator (tPA), which drives altered extracellular matrix (ECM) degradation in placental decidua. This culminates into functional dysregulation in feto-placental axis, leading to reduced size of fetus and placenta. Therefore, this study provides novel insights of a S100a10-Annexin A2 axis associated mechanism for alteration of ECM remodelling in placental decidua due to BPA exposure, which may lead to toxicity related adverse pregnancy outcome.

Introduction: Central regulation of energy homeostasis is essential for balancing lipid storage and reproductive investment; however, the hypothalamic factors governing this trade-off remain incompletely defined in birds. Neurosecretory protein GM (NPGM), an 83-amino acid hypothalamic factor, was investigated for its role in energy allocation during sexual maturation in Japanese quail (Coturnix japonica). Methods: Male and female quails at the onset of sexual maturation received chronic intracerebroventricular administration of NPGM for 13 days via osmotic pumps, during which their body mass, food intake, and water intake were monitored daily. At the endpoint, peripheral tissue and muscle masses, serum metabolite levels (glucose, fatty acids, triglycerides, testosterone, and 17{beta}-estradiol), hepatic triglyceride content, and gene expression profiles of hypothalamic feeding/reproductive genes and hepatic/adipose lipid metabolic genes were evaluated. Results: NPGM increased subcutaneous and abdominal fat in both sexes and was associated with suppressed gonadal maturation, as indicated by reduced testicular mass relative to body mass and lower testosterone levels in males, as well as a trend toward reduced ovarian mass and lower 17{beta}-estradiol levels in females. Sex-dependent metabolic phenotypes emerged: males exhibited increased body mass gain, hyperphagia, elevated water intake, enlarged liver, pancreas, and heart, higher serum and hepatic triglyceride levels, increased hepatic SCD1 expression, and reduced hepatic CGI-58, PPAR{gamma}, SLC2A2, and CD36. In contrast, females showed fat accumulation without hyperphagia or hepatic triglyceride elevation, accompanied by reduced hepatic VTG2 and APOV1 and decreased adipose ATGL, LPL, and FATP. Hypothalamic AGRP expression decreased in males, whereas both NPY and AGRP decreased in females. Discussion: These findings demonstrate that central NPGM shifts energy allocation from reproduction toward lipid storage through sex-dependent endocrine and metabolic mechanisms, identifying NPGM as a neuroendocrine regulator of energy allocation during sexual maturation in Japanese quails.

Parkinson's disease (PD) is a progressive neurodegenerative condition characterized by the loss of dopaminergic (DA) neurons and alpha-synuclein aggregation, with ferroptosis playing a critical pathological role. This study investigated the neuroprotective potential of Kocuria rhizophila strain CDMP12, a marine bacterium isolated from the Gulf of Mannar, India, using Caenorhabditis elegans models of PD. Dietary supplementation with K. rhizophila (CDMP12) significantly preserved DA neuron structure, rescued neuro-sensory and motor deficits, and attenuated both alpha-synuclein expression in the C. elegans models. Transcriptomic and qRT-PCR analyses revealed that CDMP12 systematically suppressed ferroptosis by significantly downregulating iron and lipid regulatory genes such as smf-3, ftn-1, and acs-4, while upregulating the protective antioxidant gene gpx-1. Furthermore, BODIPY staining demonstrated that CDMP12 treatment markedly reduced lipid peroxidation, lowering the oxidized-to-non-oxidized lipid ratio in PD worms. Collectively, these findings identify K. rhizophila (CDMP12) as a promising marine-derived neuroprotective candidate that mitigates PD-associated pathology, accompanied by reduced alpha-synuclein burden, preservation of DA neuronal function, and attenuation of ferroptosis-associated molecular and lipid peroxidation signatures.

Background & aims: The human gut microbiota plays a key role in health. Factors shaping its composition and diversity have been widely studied during infancy and adulthood, but far less in adolescence, despite this being a key developmental stage. We examined the potential associations between the gut microbiota of adolescents and 82 variables measured from pregnancy to adolescence. Methods: Stool samples were collected from 366 adolescents (age range: 13-16 years) from two INMA cohorts (Spain), while a range of variables, including diet, lifestyle, sociodemographic factors, health status, antibiotic use, vaccination, COVID-19, anthropometrics, and pubertal development, were collected from pregnancy to adolescence. The gut microbiota was characterized using 16S rRNA gene sequencing and assessed using - and {beta}-diversity indices and individual taxa. Associations with the study variables were evaluated using linear models, permutational multivariate analysis of variance (PERMANOVA), and Microbiome Multivariable Association with Linear Models (MaAsLin2). Results: During pregnancy, vegetable intake was positively associated with -diversity and with both {beta}-diversity and the abundance of four genera (three inverse associations and one positive association). Legume intake was also inversely associated with two genera. During adolescence, cereal and pasta intake was positively associated with -diversity and {beta}-diversity, and was inversely associated with Bacteroides, whereas fish and seafood intake was inversely associated with -diversity. Other relevant variables measured during pregnancy and at birth, such as biological sex, parental social class, and maternal education, and during adolescence, such as antibiotic use, body mass index, and pubertal status, were also associated with {beta}-diversity indices and different taxa in both directions. Conclusions: Diet during pregnancy and adolescence, together with some anthropometric, clinical, and biological factors, appeared to play an important role in shaping the composition and diversity of the gut microbiota in this population of adolescents.

Kakadu plum (Terminalia ferdinandiana), an Australian native fruit, is among the richest known dietary sources of vitamin C and hydrolysable tannins, yet its capacity to protect the intestinal epithelium against oxidative stress remains largely unexplored. This study optimised the extraction of bioactive compounds from freeze-dried Kakadu plum powder and evaluated their antioxidant activity using both chemical and cellular antioxidant in vitro assay. Phenolic compounds were extracted using three solvents (water, 80% ethanol, and 80% methanol) combined with shaking, ultrasound, or microwave assistance. Solvent, rather than processing technique, was the dominant determinant of antioxidant capacity: ethanol and methanol maximised total phenolic content, total flavonoid content, and DPPH radical-scavenging activity, whereas water extracts showed the highest ferric-reducing antioxidant power. Twenty-four phenolic compounds identified by HPLC-ESI-QTOF-MS/MS were mapped by network pharmacology to nine core oxidative-stress targets, and cross-species molecular docking predicted conserved binding of key phenolics to canine orthologs of PTGS2 and MMP2. In an H2O2-induced oxidative-stress in vitro cell model using canine small intestinal epithelial cells, both water (less than 25 ug/mL) and ethanol (less than 250 ug/mL) extracts significantly suppressed intracellular reactive oxygen species (ROS) in a dose-dependent manner, with the ethanol extract effective across a wider concentration range. This work demonstrated that Kakadu plum extract could be a promising natural, multi-target antioxidant ingredient for canine intestinal health, and provided a reference for future in vivo research.

Gut-brain communication has emerged as a rapidly expanding field of research, with recent electrophysiological studies revealing rhythmic gut-brain coupling between gastric activity and brain oscillations in humans. Gut motility is a key determinant of gastrointestinal function, but it remains unclear whether individual differences in gut motility reflected by weekly bowel movements (e.g., defecation frequency) are associated with differences in gut-brain coupling. Here, we address this question by examining women with self-reported daily bowel movements (N = 38) and women with less frequent bowel movements (N = 38). We recorded simultaneous electroencephalography (EEG) and electrogastrography (EGG) at fasting state, performed cognitive assessments, and analysed faecal short-chain fatty acids (SCFAs) as markers of colonic fermentation. In a subset of participants, EEG-EGG coupling was assessed twice over an interval of at least eight weeks to assess test-retest reliability. In this group, EEG-EGG coupling showed moderate test-retest reliability (Intraclass Correlation Coefficient (ICC) = 0.50). When comparing the two groups of women, the phase-amplitude coupling (PAC) analysis between EEG and EGG signals revealed a significantly stronger gut-brain coupling in women with daily bowel movements compared to women with less frequent bowel movements (p = 0.03). We additionally found that women with daily bowel movements made less errors in the cognitive tasks and had higher levels of faecal SCFAs. A path analysis suggested that bowel movements significantly affect gut-brain phase-amplitude coupling through faecal SCFAs. However, neither faecal SCFAs nor phase-amplitude coupling significantly predicted cognitive performance, suggesting the existence of alternative pathways for the association between bowel movements and cognitive performance. Together, our findings suggest that the strength of gut-brain coupling is associated with bowel movements and cognitive performance, making EEG-EGG coupling a promising marker of human gut-brain interactions.

Nitrogenous waste excretion is essential for all developmental stages of fish. Embryonic fish excrete urea, transitioning to cutaneous and later branchial ammonia excretion. In zebrafish, ammonia excretion involves rhesus glycoproteins Rhbg and Rhcgb in keratinocytes and ionocytes, but the developmental moment they appear in the gill remains unclear. Potential redundancy between Rhbg and Rhcgb in ammonia excretion is also not fully investigated, nor is the difference in response to low pH. We hypothesized that rhesus glycoproteins are partially redundant, and that they differ in their response to low pH as ammonia excretion enables ionocytes to exchange Na+ and H+ (Rh-NHE-metabolon). We predicted that a loss of rhbg or rhcgb induces compensatory responses. We characterized the transition from urea to branchial ammonia excretion from 0 to 8 days-post fertilization (dpf) and the response to pH 5.0 on the expression and localization of rhesus glycoproteins in control zebrafish and rhbg or rhcgb-crispants. Effects of high external ammonia (HEA, 500 M NH4Cl) and 10 mM HEPES-buffering were further characterized in rhcgb-crispants. Rhag and Rhbg appeared in the gill at 5 dpf, while Rhcgb appeared at 6 dpf. A loss of rhbg or rhcgb did not impact baseline N-excretion, illustrating that zebrafish can maintain ammonia excretion without the full complement of rhesus glycoproteins. We observed no compensatory increase in rhesus glycoproteins, but expression of the transporter hippocampus-abundant transcript 1b increased. HEA-exposed rhcgb-crispants switched to urea as primary nitrogen waste. Together, these findings underline the plasticity of the larval in dealing with nitrogenous waste.