Normal Drinking Water Research Articles

Metabolomic insights into the beneficial effects of dietary purple potato on DSS-induced murine colon colitis

Inflammatory bowel disease (IBD) is characterized by disturbances in intestinal flora and metabolomic profiles. Building on previous findings that purple potato (PP) supplementation suppresses inflammation and restores intestinal barrier in dextran sulfate sodium (DSS)-treated mice, this study examined changes in the fecal metabolome in the same cohort. Mice were fed 0 % or 10 % PP for 7 weeks, with DSS administered in the fifth week of dietary treatment followed by one-week normal drinking water recovery. A total of 68 metabolites, including amino acids, fatty acids, organic acids, carbohydrates, and polyamines, were significantly altered across the groups. PP supplementation reversed changes in 13 of the 25 DSS-altered metabolites. Six microbial metabolites derived from PP polyphenols were identified and positively correlated with M2 macrophage polarization. Data suggest that dietary PP is effective in reducing inflammation and improves gut metabolites in DSS-induced colitis mice, offering a potential dietary therapy for managing IBD.

Mitochondrial antioxidant SkQ1 attenuates C26 cancer-induced muscle wasting in males and improves muscle contractility in female tumor-bearing mice.

Mitochondrial dysfunction is a hallmark of cancer cachexia (CC). Mitochondrial reactive oxygen species (ROS) are elevated in muscle shortly after tumor onset. Targeting mitochondrial ROS may be a viable option to prevent CC. The aim of this study was to evaluate the efficacy of a mitochondria-targeted antioxidant, SkQ1, to mitigate CC in both biological sexes. Male and female Balb/c mice were injected bilaterally with colon 26 adenocarcinoma (C26) cells (total 1 × 106 cells) or PBS (equal volume control). SkQ1 was dissolved in drinking water (∼250 nmol/kg body wt/day) and administered to mice beginning 7 days following tumor induction, whereas control groups consumed normal drinking water. In vivo muscle contractility of dorsiflexors, deuterium oxide-based protein synthesis, mitochondrial respiration and mRNA content of mitochondrial, protein turnover, and calcium channel-related markers were assessed at endpoint (25 days following tumor induction). Two-way ANOVAs, followed by Tukey's post hoc test when interactions were significant (P ≤ 0.05), were performed. SkQ1 attenuated cancer-induced atrophy, promoted protein synthesis, and abated Redd1 and Atrogin induction in gastrocnemius of C26 male mice. In female mice, SkQ1 decreased muscle mass and increased catabolic signaling in the plantaris of tumor-bearing mice, as well as reduced mitochondrial oxygen consumption, regardless of tumor. However, in females, SkQ1 enhanced muscle contractility of the dorsiflexors with concurrent induction of Ryr1, Serca1, and Serca2a in TA. In conclusion, the mitochondria-targeted antioxidant SkQ1 may attenuate CC-induced muscle loss in males, while improving muscle contractile function in tumor-bearing female mice, suggesting sexual dimorphism in the effects of this mitochondrial therapy in CC.NEW & NOTEWORTHY Herein, we assess the efficacy of the mitochondria-targeted antioxidant SkQ1 to mitigate cancer cachexia (CC) in both biological sexes. We demonstrate that SkQ1 administration attenuates muscle wasting induced by C26 tumors in male, but not female, mice. Conversely, we identify that in females, SkQ1 improves muscle contractility. These phenotypic adaptations to SkQ1 are aligned with respective responses in muscle protein synthesis, mitochondrial respiration, and mRNA content of protein turnover, as well as mitochondrial and calcium handling-related markers.

Ameliorative Efficacy of <i>Moringa oleifera</i> Lam. against the Sodium Fluoride Induced Nephrotoxicity in Wistar Rat

A study was conducted to evaluate the protective efficacy of a methanolic leaf extract of Moringa oleifera L. (MO) for the remediation of nephrotoxicity induced by exposure to a significant groundwater pollutant, fluoride. Wistar albino male rats weighing 150–200 g were divided into five groups, each containing five animals. The control group received normal drinking water, and Group I received sodium fluoride in drinking water (20 mg/l daily) for 90 days. Group II received M. oleifera extract (250 mg/l) for 90 days. Group III received sodium fluoride 20 mg/l along with a low dose of M. oleifera extract (250 mg/l) for 90 days, and Group IV received sodium fluoride 20 mg/l along with a high dose of M. oleifera extract (500 mg/l) for 90 days. After 90 days, the kidneys and blood samples were obtained to evaluate their histological characteristics and assess the levels of oxidative stress and various enzyme parameters. The current investigation showed that sodium fluoride has a notable impact on renal function by elevating levels of creatinine, Blood Urea Nitrogen (BUN), urea, as well as Serum Glutamic Oxaloacetic Transaminase (SGOT), Serum Glutamate Pyruvate Transaminase (SGPT), and Alkaline Phosphatase (ALP) indicators. Further exposure resulted in a decrease in Superoxide Dismutase (SOD) and Catalase (CAT) levels, whereas Lipid Peroxidation (LPO) activity increased in kidney tissue. The histopathological examination of the kidney revealed glomerular enlargement, degenerative changes in Bowman’s capsule, and alterations in the tubular portion of the glomerulus. The study’s findings indicate that the administration of M. oleifera extract had a notable impact on the renal function and antioxidant status of rats subjected to sodium fluoride treatment. The administration of M. oleifera extract demonstrated a significant ability to mitigate the nephrotoxic effects induced by sodium fluoride in rats, mainly when both substances were concurrently administered. M. oleifera extract may also provide hepatoprotective effects by modifying membrane permeability and blocking enzymes from entering the serum.

Chronic N-acetyl cysteine treatment does not improve respiratory system performance in the mdx mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is characterised by respiratory muscle injury, inflammation, fibrosis and weakness, ultimately culminating in respiratory failure. The dystrophin-deficient mouse model of DMD (mdx) shows evidence of respiratory muscle remodelling and dysfunction contributing to impaired respiratory system performance. The antioxidant N-acetylcysteine (NAC) has been shown to exert anti-inflammatory and anti-fibrotic effects leading to improved respiratory muscle performance in a range of animal models of muscle dysfunction, including mdx mice, following short-term administration (2weeks). We sought to build on previous work by exploring the effects of chronic NAC administration (3months) on respiratory system performance in mdx mice. One-month-old male mdx mice were randomised to receive normal drinking water (n=30) or 1% NAC in the drinking water (n=30) for 3months. At 4months of age, we assessed breathing in conscious mice by plethysmography followed by ex vivo assessment of diaphragm force-generating capacity. Additionally, diaphragm histology was performed. In separate studies, in anaesthetised mice, respiratory electromyogram (EMG) activity and inspiratory pressure across a range of behaviours were determined, including assessment of peak inspiratory pressure-generating capacity. NAC treatment did not affect force-generating capacity of the mdx diaphragm. Collagen content and immune cell infiltration were unchanged in mdx + NAC compared with mdx diaphragms. Additionally, there was no significant effect of NAC on breathing, ventilatory responsiveness, inspiratory EMG activity or inspiratory pressure across the range of behaviours from basal conditions to peak system performance. We conclude that chronic NAC treatment has no apparent beneficial effects on respiratory system performance in the mdx mouse model of DMD suggesting limited potential of NAC treatment alone for human DMD.

ELUCIDATING THE EFFECTS OF FORMALIN AS FOOD PRESERVATIVE ON GROSS AND HISTOARCHITECTURE OF VITAL ORGANS OF SWISS ALBINO MICE

Formalin usage as food preservative in Bangladesh has dramatically increased recently, especially in fish, fruits, and many other food products, putting the vast majority of people in serious health risk. With this background, the study was aimed to detect the alterations of gross structure and histoarchitecture of vital organs in Swiss albino mice due to the consumption of formalin. Mice were divided into five groups at the age of four weeks. Normal drinking water was supplied to the control group of mice. Mice of group 1, 2, 3 and 4 were supplied normal drinking water mixed with formalin at 1%, 5%, 10% and 20% concentrations, respectively up to week 24. Mice treated with varying concentrations of formalin for 24 weeks was assessed for the weight change of their liver, heart, kidney and lung weights. Investigations were also conducted on histological alterations in the liver, heart, kidney and lung. After 24 weeks of treatment of mice with different concentrations of formalin, the weight of all four major organs (liver, heart, kidney and lung) was decreased in all formalin-treated mice in comparison to control mice. Petechial hemorrhages, congestion, necrosis, and parenchymatous cell degeneration have been found in liver. Kidney sections have shown decreased Bowman’s space, hypertrophied glomerulus and tubular dilatation. Leucocyte infiltration, necrosis, and rupture of connective tissue have been seen in the heart sections. Lung sections have shown pulmonary alterations, including hemorrhages, thicker alveolar walls, and inflammatory cells. It was, concluded that formalin has adverse effects on vital organs of Swiss Albino mice.

Empagliflozin and pirfenidone confer renoprotection through suppression of glycogen synthase kinase-3β and promotion of tubular regeneration in rats with induced metabolic syndrome

Metabolic syndrome (MetS) is largely coupled with chronic kidney disease (CKD). Glycogen synthase kinase-3β (GSK-3β) pathway drives tubular injury in animal models of acute kidney injury; but its contribution in CKD is still elusive. This study investigated the effect empagliflozin and/or pirfenidone against MetS-induced kidney dysfunction, and to clarify additional underpinning mechanisms particularly the GSK-3β signaling pathway. Adult male rats received 10%w/v fructose in drinking water for 20 weeks to develop MetS, then treated with either drug vehicle, empagliflozin (30 mg/kg/day) and/or pirfenidone (100 mg/kg/day) via oral gavage for subsequent 4 weeks, concurrently with the high dietary fructose. Age-matched rats receiving normal drinking water were used as controls. After 24 weeks, blood and kidneys were harvested for subsequent analyses. Rats with MetS showed signs of kidney dysfunction, structural changes and interstitial fibrosis. Activation of GSK-3β, decreased cyclinD1 expression and enhanced apoptotic signaling were found in kidneys of MetS rats. There was abundant alpha-smooth muscle actin (α-SMA) expression along with up-regulation of TGF-β1/Smad3 in kidneys of MetS rats. These derangements were almost alleviated by empagliflozin or pirfenidone, with evidence that the combined therapy was more effective than either individual drug. This study emphasizes a novel mechanism underpinning the beneficial effects of empagliflozin and pirfenidone on kidney dysfunction associated with MetS through targeting GSK-3β signaling which can mediate the regenerative capacity, anti-apoptotic effects and anti-fibrotic properties of such drugs. These findings recommend the possibility of using empagliflozin and pirfenidone as promising therapies for management of CKD in patients with MetS.

Sodium dichloroisocyanurate: improving broiler health by reducing harmful microbial levels in the waterline.

Sodium dichloroisocyanurate (NaDCC) is commonly used for treating drinking water, industrial water, and wastewater. This study aimed to investigate the potential effects of NaDCC-treated waterline drinking water on the growth of AA+ broilers by reducing microbial levels in the waterline. A total of 480 healthy 1-day-old AA+ broilers (46.77 ± 0.50 g) were selected for the experiment and randomly divided into four groups with six replicates of 20 birds each. The control group received regular drinking water, while the test groups received drinking water with NaDCC concentrations of 10, 30, and 50 mg/L. The test groups consumed the treated water on specific days throughout the 42-day experimental period. Results showed that NaDCC treatment significantly reduced the levels of E. coli, Salmonella, S. aureus and Moulds in the drinking water at the waterline (p < 0.05). Drinking water with NaDCC also led to reduced broiler fecal emissions of NH3 and H2S, as well as reduced counts of E. coli, Salmonella, S. aureus and Moulds (p < 0.05), particularly at 30 mg/L and 50 mg/L concentrations. Broilers consuming NaDCC at 50 mg/L exhibited a significant increase in ADG from days 1-42 (p < 0.05). The levels of E. coli, Salmonella, S. aureus and Moulds in the drinking water at the waterline were significantly and positively correlated with the bacterial count in the feces (p < 0.05, R > 0.6). Additionally, bacterial levels in drinking water and broiler feces were negatively correlated with broiler production performance indicators, including ADG, ADFI, F/G and AWC. In conclusion, NaDCC can indirectly enhance broiler performance by reducing the levels of harmful bacteria in the waterline without affecting normal drinking water. The addition of 30 mg/L or 50 mg/L of NaDCC to the waterline in poultry production can effectively control harmful microorganisms and improve poultry health. Based on the experiment's results, it is recommended to preferentially use 30 mg/L NaDCC in the waterline to reduce farming costs.

Zinc Ameliorates Cadmium-Induced Immunotoxicity by Modulating Splenic Immunosuppressive Indoleamine 2,3-Dioxygenase Activity, Hematological Indices, and CD4+ T Cells via Inhibition of Cadmium Uptake in Male Wistar Rats.

Cadmium (Cd)-induced immunotoxicity has become a matter of public health concern owing to its prevalence in the environment consequently, great potential for human exposure. Zinc (Zn) has been known to possess antioxidant, anti-inflammatory, and immune-boosting properties. However, the ameliorating influence of Zn against Cd-induced immunotoxicity connecting the IDO pathway is lacking. Adult male Wistar rats were exposed to normal drinking water with no metal contaminants (group 1), group 2 received drinking water containing 200 μg/L of Cd, group 3 received drinking water containing 200 μg/L of Zn, and group 4 received Cd and Zn as above in drinking water for 42 days. Cd exposure alone significantly triggered the splenic oxidative-inflammatory stress, increased activities of immunosuppressive tryptophan 2,3-dioxygenase (TDO), indoleamine 2,3-dioxygenases (IDO) activities/protein expression, and decreased CD4+ T cell count, and a corresponding increase in the serum kynurenine concentration, as well as alterations in the hematological parameters and histologic structure when compared with the control (p < 0.05). Zn alone did not have any effect relative to the control group while co-exposure significantly (p < 0.05) assuaged the Cd-induced alterations in the studied parameters relative to the control. Cd-induced modifications in IDO 1 protein expression, IDO/TDO activities, oxidative-inflammatory stress, hematological parameters/CD4+ T cell, and histological structure in the spleen of rats within the time course of the investigation were prevented by Zn co-exposure via inhibition of Cd uptake.

1542-P: Commercially Available Aspartame-Based Sweetener Damages Morphology of Major Organs Both in Normal and Type 2 Diabetic Rats

Non-nutritive sweeteners (NNS) such as aspartame have become highly popular as sugar substitutes for diabetics. Majority of studies have reported controversial results and used aspartame in its pure form, which is not consumed by people practically. Hence, the present study was aimed at investigating the effects of commercially available aspartame-based sweetener on diabetes related parameters and its associated complications in both normal and type 2 diabetic rats. Seven-week-old male Sprague-Dawley rats were randomly divided into four groups, namely: Normal Control (NC), Toxicological Aspartame (TASP), Diabetic Control (DBC) and Diabetic Aspartame (DASP). Type 2 diabetes was induced experimentally in the DBC and DASP groups only and animals with non-fasting blood glucose levels &amp;gt;300 mg/dL were considered as diabetic. During 13 weeks experimental period, the control groups received normal drinking water, whilst TASP and DASP groups were given aspartame-containing NNS solutions, ad libitum, at concentrations equivalent to the sweetness of 20% and 10% sucrose solution, respectively. Treatment with toxicological aspartame resulted in significantly reduced serum fructosamine and creatinine levels in normal rats, when brain, heart, liver, kidney and pancreatic tissue morphologies were significantly damaged in both normal and diabetic rats. The data of this study suggest that although aspartame-based NNS may not have higher degree of detrimental effects on diabetes associated parameters, the significantly damaged morphology of the major organ tissues as well as deterioration of related serum biomarkers both in normal and diabetic conditions raises a concern of its use as a NNS in various food and food products. Disclosure S.N.Dlamini: None. S.Islam: None. Funding South African Sugar Association (Project 233)

A multi-targeted T-cell engagement of PD1-resistant colorectal cancer cells via delivery of glycopeptide conjugates.

e14571 Background: Glycan-based cancer immunotherapies are novel therapeutic strategies that use the pivotal role of aberrant glycosylation patterns in cancer progression. Targeting hybrid epitopes consisting of altered glycans and carrier peptides enhances the efficacy and specificity of the anti-tumor immune response. Here, we exploit ensembles of glycopeptide conjugates to steer a multi-targeted yet tumor-specific T-cell response, a technology we call GLAAD (glycopeptides with adjuvant and tumor-associated antigen delivery). By inducing T cells that recognize and eliminate tumor cells and promoting their antigenicity in a therapeutic setting, we show the potential of our GLAAD candidate GNU-101 to overcome the transient and heterogeneous responses frequently observed with traditional immunotherapies. Methods: Using the MC38 syngeneic mouse model of colorectal cancer (CRC) in a therapeutic setting, we tested whether GNU-101 reduces tumor burden when orally administered alone or combined with immune checkpoint inhibitors (ICI). On day 0, 20 C57/BL6J mice were injected subcutaneously with MC38 CRC cells in each flank. On day 6, mice were supplemented with 3% GNU-101 in the drinking water, while control mice received normal drinking water. On days 6, 9, 12, and 15, half of the mice in each group received an anti-PD1 antibody (aPD1), while the others received isotype control. On day 16, the mice were sacrificed. Results: In this therapeutic setting, a standalone therapy of GNU-101 successfully reduced tumor growth as demonstrated by a statistically significant reduction in both tumor volume and weight. As expected, the aPD1 therapy had only a non-significant effect on tumor growth. Remarkably, its efficacy was improved when combined with GNU-101, leading to a statistically significant reduction of &gt;90% in tumor volume and weight. The analysis of the immune cell population in the tumor microenvironment revealed the activation of CD8 T cells and a reduction of immunosuppressive signals, which likely mediates the anti-tumor efficacy of GNU-101. This was obvious by a higher abundance of CD8 T cells producing IFNγ and secreting granzyme B &amp; perforin and, conversely, lower amounts of Tim3+ CD8 T cells and regulatory T cells (FoxP3+ CD4 T cells) found in mice treated with GNU-101 vs control mice. Conclusions: We demonstrate the immunomodulatory and therapeutic potential of GNU-101 in a pre-clinical mouse study in which the growth of established tumors was significantly reduced, either as a standalone therapy or in combination with existing treatment. By specifically promoting anti-cancer CD8+ T cells while reducing immunosuppressive signals, this easy-to-administer glycopeptide has the potential to create the conditions for long-term cancer control and improve treatment outcomes in cancer that are frequently resistant to immunotherapy, such as many epithelial cancers.

Growth performance and cecal microbiota of broiler chicks as affected by drinking water disinfection and/or herbal extract blend supplementation

Environmental exposures during early life are important for animals' intestinal microbiota composition and their production performance. This experiment investigated the growth performance, hematology parameters, jejunal morphology, and cecal microbiota of broiler chicks as affected by exogenous factors from the aspects of drinking water quality and dietary manipulation. A total of 480-day-old broiler chicks (Arbor acre; 41.59 ± 0.88 g) were randomly assigned into 4 groups (CON, HWGM, CA, CAHWGM). Each group had 6 replicates with 20 birds per replicate. Broiler chicks in CON group were fed with basal diet and drank normal drinking water; in HWGM group were fed with basal diet supplemented with 1.5g/kg herbal extract blend (hops, grape seed, and wheat germ) and drank normal drinking water; in CA group were fed with basal diet and drank sodium dichlorocyanurate (50 mg/L) treated-drinking water; in CAHWGM group were fed with basal diet supplemented with 1.5 g/kg herbal extract blend and drank chlorinated drinking water. The experimental period was 42 d. We found that broiler chicks drank chlorinated drinking water led to an increase in body weight gain and feed efficiency during d 22 to 42 and 1 to 42, as well as a decrease in cecal Dysgonomonas and Providencia abundance. Dietary supplementation of herbal extract blend increased cecal Lactobacillus and Enterococcus abundance, whereas decreased Dysgonomonas abundance. Moreover, we observed that cecal Dysgonomonas abundance synergistically decreased by treating drinking water with sodium dichlorocyanurate and supplementing herbal extract blend to the diet. Therefore, results obtained in this study indicated that providing chlorinated drinking water is an effective strategy to improve the growth performance of broiler chicks by regulating intestinal microbiota. Additionally, dietary supplementation of herbal extract blend alone or combined with chlorinated drinking water is able to regulate cecal microbiota.

Cold Drinking Water Boosts the Cellular and Humoral Immunity in Heat-Exposed Laying Hens

This study aimed to investigate the effects of cold drinking water on cellular and humoral immunity in heat-exposed laying hens. One hundred and eight laying hens at 19 weeks old were placed into three treatments with six replicates of six hens in each group as follows: (1) hens were provided with normal drinking water (NW) under the control of thermoneutral temperature (CT: 25 ± 1 °C; CT + NW), (2) hens were provided with NW under high ambient temperature (HT: 35 ± 1 °C; HT + NW) for 8 h/d for a month, and (3) hens were treated under HT with cold drinking water (CW: 15 ± 1 °C; HT + CW) for 8 h/d for a 4-weeks. Then, the feed consumption, egg production, egg weight, feed conversion ratio, and blood immune parameters were investigated. The results showed that cold drinking water (CW) caused a significant (p < 0.05) recovery in the reduction of food intake and egg production due to heat stress; however, there was no significant effect (p > 0.05) on egg weight and feed conversion ratio. Moreover, CW significantly (p < 0.05) restored the immune-suppressing effects of heat stress on the contents of peripheral blood mononuclear cells, including B-cell (BU-Ia), helper T cell (CD4), and the ratio of helper/cytotoxic T cell (CD4/CD8). In addition, CW significantly (p < 0.05) recovered the reduction on the level of mRNA expression of interleukin-2 (IL-2) and interferon-gamma (IFN-γ), as well as significantly (p < 0.05) restored the reduction of plasma concentration of IL-2, IFN-γ and immunoglobulin G in heat-stressed laying hens. These results prove that CW increased heat dissipation and enhanced feed intake, egg production, and cellular and humoral immunity in heat-exposed laying hens.

The Amino Acid Homoarginine Inhibits Atherogenesis by Modulating T-Cell Function.

Amino acid metabolism is crucial for inflammatory processes during atherogenesis. The endogenous amino acid homoarginine is a robust biomarker for cardiovascular outcome and mortality with high levels being protective. However, the underlying mechanisms remain elusive. We investigated the effect of homoarginine supplementation on atherosclerotic plaque development with a particular focus on inflammation. Female ApoE-deficient mice were supplemented with homoarginine (14 mg/L) in drinking water starting 2 weeks before and continuing throughout a 6-week period of Western-type diet feeding. Control mice received normal drinking water. Immunohistochemistry and flow cytometry were used for plaque- and immunological phenotyping. T cells were characterized using mass spectrometry-based proteomics, by functional in vitro approaches, for example, proliferation and migration/chemotaxis assays as well as by super-resolution microscopy. Homoarginine supplementation led to a 2-fold increase in circulating homoarginine concentrations. Homoarginine-treated mice exhibited reduced atherosclerosis in the aortic root and brachiocephalic trunk. A substantial decrease in CD3+ T cells in the atherosclerotic lesions suggested a T-cell-related effect of homoarginine supplementation, which was mainly attributed to CD4+ T cells. Macrophages, dendritic cells, and B cells were not affected. CD4+ T-cell proteomics and subsequent pathway analysis together with in vitro studies demonstrated that homoarginine profoundly modulated the spatial organization of the T-cell actin cytoskeleton and increased filopodia formation via inhibition of Myh9 (myosin heavy chain 9). Further mechanistic studies revealed an inhibition of T-cell proliferation as well as a striking impairment of the migratory capacities of T cells in response to relevant chemokines by homoarginine, all of which likely contribute to its atheroprotective effects. Our study unravels a novel mechanism by which the amino acid homoarginine reduces atherosclerosis, establishing that homoarginine modulates the T-cell cytoskeleton and thereby mitigates T-cell functions important during atherogenesis. These findings provide a molecular explanation for the beneficial effects of homoarginine in atherosclerotic cardiovascular disease.

Gingerol fractions bioactivity against butanone cytotoxicity induced in newborns of mice.

Accumulating studies have demonstrated the potential activity of ginger in treating and managing several diseases but little is known about its protective effects against teratogenicity of chemical toxins. Thus, in this study, we have evaluated the protective effect of gingerol fraction (GF) against methyl ethyl ketone (MEK) induced teratogenic effects in newborns of mice. A total of 30 mature females and fifteen male mice (Mus musculus) weighing 25-30 g were included in this study. The pregnant mice were divided into three groups (10 mice each); control group (GI, mice received normal drinking water; NDW), methyl ethyl ketone (MEK) treated group (GII, received MEK at a dose of 350 mg/kg body weight in NDW), and GF treated group (GIII; mice received GF at a dose of 25 mg/kg in NDR). Histological analysis, cellular oxidative, and antioxidant enzymes, fibrosis, and apoptosis of brain, liver, and kidney tissues were estimated by histological and immunoassay techniques. In this study, the treatment of pregnant female mice with gingerol fractions (GF) at a dose of 25 mg/kg significantly protected all tissues organs of mothers and their offspring against the teratogenic effects induced by MEK at a dose of 350 mg/kg. A significant improvement in cellular antioxidant enzymes GSH, SOD, and peroxidase activities along with a reduction in the initiation of cellular oxidative free radicals (TBARS) was reported in GF treated mice compared to mice intoxicated with MEK (350 mg/kg). In addition, a significant reduction in cellular fibrosis and apoptosis was reported in all tissues of mothers and their offspring's following treatment with GF. HPLC analysis of ginger extracts estimated a set of polyphenolic compounds such [6]-gingerol, [8]-gingerol, [10]-gingerol, and [6]-shogaol which are responsible for the antioxidant, anti-fibrotic, and anti-apoptotic protective effects against teratogenic effects of MEK. Gingerol fractions (GF) at a dose of 25 mg/kg significantly protected all tissues organs of mothers and their offspring against the teratogenic effects induced by MEK at a dose of 350 mg/kg. The beneficial effects of ginger phenolic compounds; [6]-gingerol, [8]-gingerol, [10]-gingerol, and [6]-shogaol against teratogenic effects of MEK proceeded through their antioxidant, anti-fibrotic, and anti-apoptotic properties.

Abstract 5600: Glycopeptides promote anti-cancer immune response against solid tumors

Abstract Only a fraction of patients benefits from immune checkpoint inhibitors (ICI). Tumor cells exhibit a distinct glycosylation pattern and supplementation of glycans has the potential to promote response and overcome resistance to ICI, thus addressing an urgent clinical need. Using a subcutaneous tumor mouse model, we investigated whether two glycopeptide-based test items reduce tumor burden when orally administered alone or in combination with ICI. C57/BL6 mice were supplemented with 3% GNU-201 (n=10) or 3% GNU-101 (n=10) in the drinking water starting 14 days prior to tumor cell injection (d-14), while control mice (n=10) received normal drinking water. On day 0, all mice were injected subcutaneously with YUMM1.7 melanoma cells in each flank. On days 6, 9 and 12, mice in each group received anti-PD1 antibody or an isotype control. Tumor size was measured every 3rd day. On day 15, the mice were sacrificed to analyze tumor weight and tumor infiltrating immune cells. 16S rDNA sequencing of fecal samples was performed on days -14, 0 and 14. GNU-201 and GNU-101 supplementation was well tolerated, no difference in water consumption and no abnormal behavior were detected that would indicate adverse effects of the products. While anti-PD1 on its own had no effect on tumor growth, we observed reduced tumor growth in the GNU-201+anti-PD1, GNU-101 and GNU-101+anti-PD1-treated groups, resulting in a significantly reduced tumor volume in these three groups on day 15. Administration of the two test items had a clear effect on anti-tumor T cell responses. In combination with anti-PD1, GNU-201 promoted the overall abundance of T cells, and among those the proportion of CD8+ T cells and IFNγ producing CD4+ T cells. Even in absence of anti-PD1, GNU-201 promoted the number of IFNγ+ CD8+ T cells, indicating that GNU-201 did not only promote tumor infiltration but also activation of CD8+ T cells, which are known to directly kill tumor cells. Finally, GNU-201+anti-PD1 resulted in significantly increased levels of Perforin and TNFα producing CD8+ T cells. With GNU-101 there was a trend towards elevated numbers of T cells and TNFα+ CD4 T cells. In mice that received GNU-101 with anti-PD1, we observed significantly elevated levels of TNFα+ CD8+ T cells and a trend towards higher numbers of IFNγ+ CD8+ T cells. GNU-201 and GNU-101 induced a significant modulation of the gut microbiota. In particular, the products promoted the growth of Akkermansia spp., a genus previously associated with anti-tumor immunity. In conclusion, GNU-201 and GNU-101 are both able to boost an anti-tumor immune response upon PD1 inhibition and GNU-101 shows moderate anti-tumor effects on its own. The response is linked with tumor-infiltration and activation of T cells and seems to be associated with changes in the gut microbiome. This study highlights a potential therapeutic role for glycopeptides in boosting anti-tumor immunity in response to ICI therapy. Citation Format: Marianne R. Spalinger, Romain Wyss, Sara Vidal, Yong Miao, Michael Scharl, Yemi Adesokan. Glycopeptides promote anti-cancer immune response against solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5600.

Exploiting GLAAD molecules to drive an antitumor immune response in a colorectal cancer mouse model.

2565 Background: Only a minority of colorectal cancer (CRC) patients benefit from immune checkpoint inhibitors (ICI), with less than half of eligible patients responding to the treatment. Abnormal glycosylation of tumor cells is a signature of cancer development. Here, we described novel immunomodulatory molecules, named GLAAD (glycopeptides with adjuvant and tumor-associated antigen delivery). We hypothesized that GLAAD, by sharing similarities with aberrant glycans expressed in many cancers, may be a safe and effective treatment strategy to promote response and overcome resistance to ICI, thus addressing an urgent clinical need. Using a CRC mouse model, we tested whether two GLAAD candidates reduce tumor burden when orally administered alone or in combination with ICI. Methods: C57/BL6J mice were supplemented with 3% GNU-201 or 3% GNU-101 in the drinking water starting 14 days prior to tumor cell injection (d-14), while control mice received normal drinking water. On day 0, all mice were injected subcutaneously with MC38 CRC cells in each flank. On days 6, 9, 12, and 15, mice in each group received anti-PD1 antibody (aPD1) or an isotype control. Tumor size was measured every 3rd day. On day 18, the mice were sacrificed. Results: The aPD1 therapy alone showed a limited anti-tumor efficacy overall. Groups treated with GNU-201, GNU-101, GNU-201+aPD1 or GNU-101+aPD1 exhibited reduced growth, resulting in a significantly reduced tumor volume vs control on day 18. In contrast, GLAAD candidates alone significantly reduced tumor volume compared with control and led to a numerically larger decrease than aPD1 treatment alone, suggesting a higher efficacy. When analyzing tumor infiltrating myeloid cells on day 18, no differences were seen for neutrophils or dendritic cells but the proportion of macrophages was decreased in mice receiving GNU-201 or GNU-101. GNU-101 resulted in reduced serum levels of IL-10, a molecule participating in the suppression of antitumor immune responses. The tested GLAAD candidates stimulated robust anti-tumor T cell responses. GNU-201 and GNU-101 supplementation alone or in combination with aPD1 increased the proportion of T cells and among those CD8+ T cells, IFNγ producing CD4+ T cells, IFNγ+ and perforin+ CD8+ T cells. GNU-101 and GNU-201 elicited memory T cell responses after priming of the mice, measured as the ex vivo recall response (CD69 expression) of CD8+ T cells isolated from the tumor. Conclusions: GNU-201 and GNU-101 as standalone treatment induce a strong anti-tumor response that is as good as or even better than aPD1 alone. The therapeutic efficacy is associated with increased tumor infiltration and activation of T cells, in particular CD8+ T cells, highlighting the role of these GLAAD candidates as potent adjuvants in supporting specific tumor-associated antigen recognition by the immune system.

Neonatal Oral Administration of Chrysin Prevents Long-Term Development of Non-Alcoholic Fatty Liver Disease in a Sexually Dimorphic Manner in Fructose Nurtured Sprague Dawley Rats.

High-fructose diets are linked with the development of non-alcoholic fatty liver disease (NAFLD), the management of which is a burden to society. Interventions with phytochemicals in the early postnatal period may prevent fructose-induced NAFLD later in adulthood. We investigated the protective potential of chrysin against fructose-induced NAFLD. Four-day-old male and female suckling Sprague Dawley rats (N = 112) were randomly grouped and orally gavaged daily with distilled water (negative Control-Cn + W), chrysin(Chr-100 mg/kg), fructose-solution (Fr-20% w/v), and Chr + Fr between postnatal day (PND) 4 and 21 and then weaned onto normal rat chow and plain drinking water to PND 55. From PND 56 to 130, half of the rats continued on plain water, and the rest had Fr as drinking fluid. Terminally, the liver tissue was collected, and the lipid content was determined and histologically assessed for NAFLD. Dietary Fr induced an increased hepatic lipid content (p = 0.0001 vs. Cn + W) both sexes, and it was only attenuated by neonatal Chr in female rats (p < 0.05). Histologically, there was increased microvesicular steatosis (p = 0.0001 vs. Cn + W) in both sexes, and it was prevented by neonatal Chr (p > 0.05). Fr caused macrovesicular steatosis (p = 0.01 vs. Cn + W) in females only, and chrysin did not prevent it (p > 0.05). Fr induced hepatocellular hypertrophy, and inflammation was observed in females only (p = 0.01 vs. Cn + W), and this was prevented by Chr (p > 0.05). The collagen area fraction was increased by Fr (p = 0.02 (males) and p = 0.04 (females) vs. Cn + W, respectively; however, chrysin did not prevent this (p > 0.05). Neonatal chrysin prevented some of the deleterious effects of the high-fructose diet on the liver, suggesting that chrysin should be further explored as a strategic prophylactic neonatal intervention against high-fructose-diet-induced NAFLD.

Resilience and the Gut Microbiome: Insights from Chronically Socially Stressed Wild-Type Mice.

The microbiome is an important player within physiological homeostasis of the body but also in pathophysiological derailments. Chronic social stress is a challenge to the organism, which results in psychological illnesses such as depression in some individuals and can be counterbalanced by others, namely resilient individuals. In this study, we wanted to elucidate the potential contribution of the microbiome to promote resilience. Male mice were subjected to the classical chronic social defeat paradigm. Defeated or undefeated mice were either controls (receiving normal drinking water) or pre-treated with antibiotics or probiotics. Following social defeat, resilient behavior was assessed by means of the social interaction test. Neither depletion nor probiotic-shifted alteration of the microbiome influenced stress-associated behavioral outcomes. Nevertheless, clear changes in microbiota composition due to the defeat stress were observed such as elevated Bacteroides spp. This stress-induced increase in Bacteroides in male mice could be confirmed in a related social stress paradigm (instable social hierarchy) in females. This indicates that while manipulation of the microbiome via the antibiotics- and probiotics-treatment regime used here has no direct impact on modulating individual stress susceptibility in rodents, it clearly affects the microbiome in the second line and in a sex-independent manner regarding Bacteroides.

Isotopically anomalous metabolic oxygen in marine vertebrates as physiology and atmospheric proxy

Triple oxygen isotope data of tooth enamel (phosphate) from 16 modern marine vertebrates (sharks, mammals) from different marine habitats are presented. Marine mammal data show a clear signature of isotopically anomalous metabolic oxygen in their bioapatite. This is related to a low flux ratio of isotopically normal drinking and food water to anomalous metabolic oxygen. Traces of anomalous metabolic oxygen are also observed in shark bioapatite. Sharks and mammals have overlapping 18O/16O ratios, but distinct 17O signatures. Our new data suggest that triple oxygen isotopes are a valuable new tracer to assess the physiology of extant and extinct marine vertebrates and may provide a new proxy for past atmospheric CO2 mixing ratios and global primary biosphere production.

Effects of three probiotics and their interactions on the growth performance of and nutrient absorption in broilers.

The purpose of this study was to investigate the effects of three probiotics and their interactions on growth performance, intestinal digestion and absorption, and nutrient transporters in broilers. A total of 350 one-day-old male Arbor Acres broilers were randomly divided into seven groups: the control group (broilers receiving normal drinking water), groups P1, P2 and P3 (broilers receiving drinking water with 1% Lactobacillus casei, Lactobacillus acidophilus and Bifidobacterium lactis , respectively) and groups CP1, CP2 and CP3 (broilers receiving drinking water with a 1% compound probiotic mixture in 2:1:1, 1:2:1, 1:1:2 ratios, respectively). The feeding period was divided into two experimental periods: 1∼21 days and 22∼42 days. Compared to those in the control group, the broiler slaughter indexes and average daily feed intakes in the probiotics groups were not significantly different (P > 0.05), but the villus height in the small intestine increased significantly, and the crypt depth decreased significantly (P < 0.05). In the 1- to 21-day, experimental period, the broiler average daily gains in groups CP2 and CP3 were significantly greater than that in the control group. Amylase, lipase, and trypsin activities in the jejunum in groups CP and P3 increased significantly. GLUT2 mRNA expression in the probiotics group was significantly incresaed compared with that in the control group (P < 0.05). In the 22- to 42-day period, the average daily gain in the CP group was significantly greater than that in the control group. Amylase activity in the CP2 group, and lipase and trypsin activities in the CP, P1 and P3 groups increased significantly. The GLUT2 mRNA expression in the CP group increased significantly (P < 0.05). In summary, three probiotics and their interactions improved the digestibility and absorption of nutrients by increasing the activities of digestive enzymes, improving the morphology of the digestive tract, and upregulating the expression of GLUT2 mRNA in the intestinalcell membrane to improve the production performance in broilers.