Chickpea (Cicer arietinum) protein hydrolysate ameliorates the metabolic effect of feeding a high-fat-diet-induced obesity in mice by modulating inflammation

Obesity is a growing epidemic in the United States with over 40% of the country suffering from this disease. Obesity increases the risk of many chronic conditions,such as type 2 diabetes (T2D). Foods that are high in nutrients and low in calories, such as chickpeas, can help treat obesity. While whole chickpeas assist in weight loss, chickpea protein can also aid in managing T2D as it contains several bioactive peptides that might influence glucose absorption. The aim of this study was to evaluate the efficacy of a chickpea protein hydrolysate (CPH) to ameliorate markers of obesity in a 16-week mouse model of metabolic dysfunction when fed a high fat diet (HFD). Additionally, liver tissue was analyzed for evidence of non alcoholic fatty liver disease (NAFLD). CPH was produced by an alcalase and Flavourzyme®️ dual-system. The hydrolysate was characterized by LC-MSMS to detect for anti-diabetic peptides. Fifty-two six-week-old male C57BL/6J mice (n = 13/group) were randomized: standard diet (SD), HFD, HFD + 400 mg CPH/kg body weight (BW) (400 CPH), and HFD + 800 mg CPH/kg BW (800 CPH). CPH was administered daily to the treatment groups for the duration of the study. At the end of the study, plasma was analyzed for concentration of adiponectin, resistin, insulin, triglycerides, total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, and inflammatory proteins. Feces were also collected and measured for amino acid and fatty acid concentrations. Adipose tissue underwent histological analysisfor adipocyte area and macrophage clustering. Liver was scored for NAFLD progression. Genes related to insulin resistance were also analyzed in adipose and liver. Three peptides in the hydrolysate were identified having angiotensin-converting enzyme (ACE)-inhibitory and dipeptidyl peptidase-IV (DPP-IV) inhibitory properties. After the 16-week study, weight gain for 800 CPH was lower (19.2%, p 2) of genes related to carbohydrate (Gys1, Pck1, Pdk2) and lipid (Fasn, Pparg) metabolism compared to HFD control, while 800 CPH had upregulation of genes for inflammation (Il1β and Il18R1) and carbohydrate metabolism (Hk2) compared to HFD control. In liver, SD control had upregulation of genes for lipid metabolism (Acaca, Acacb, Acsl4, Cebpa, Lepr) compared HFD control while 400 CPH had downregulated genes related to adipokine (Tnfrsf1b) and insulin signaling (Pik3r1, Ikbkb, Map2k1) compared to HFD control.Submission published under a 24 month embargo labeled 'U of I Access', the embargo will last until 2024-08-01The student, Casey Radlowski, accepted the attached license on 2022-07-20 at 10:51.The student, Casey Radlowski, submitted this Thesis for approval on 2022-07-20 at 11:00.This Thesis was approved for publication on 2022-07-21 at 11:15.DSpace SAF Submission Ingestion Package generated from Vireo submission #18389 on 2022-11-15 at 21:40:3

Early nutrition affects intestinal development and immune response in the neonatal piglet

Breast milk is the optimal form of nutrition for the human infant. In the U.S., 77% of women initiate breastfeeding immediately after birth, but by 6 months post-partum, only 43% are still breastfeeding with only 13% being exclusively breastfed (CDC, 2009). The remaining 30% of mothers are using formula supplemented with breast milk or “combined feeding” (CF) (CDC, 2009). Many infants who are receiving formula as their primary of source of nutrition are deprived of beneficial compounds naturally existing in human milk. One of the groups of bioactive components is the human milk oligosaccharides (HMO). This complex group of structures has many beneficial effects for the infant including promoting the growth of Bifidobacteria, acting as anti-adhesive and anti-microbial factors against pathogens, modulating immune development, and perhaps even providing nutrients for brain development (Bode, 2012). Prebiotics are routinely added to infant formula to mimic the actions of HMOs. In addition to increasing rates of formula feeding, the proportion of infants being born by cesarean section is also on the rise with rates being approximately 30% in the U.S. (Hamilton BE, 2009; Betrán AP, 2007; Mayor S, 2005). Route of delivery can influence colonization of intestinal microbiota. Vaginally-delivered (VD) infants acquire microbes from their mother’s vaginal canal and perineum, while cesarean-derived infants obtain more microbes from the environment, including the mother’s skin (Biasucci et al, 2008, Dominguez-Bello et al, 2010). The microbes that colonize the newborn intestine influence other functions of the gut, including mucosal immune development (Mshviladadze and Neu, 2010). Herein, we have three aims to investigate the effects of these early life events on neonatal intestinal, microbial, and immune development. Our first aim was to investigate the effect of route of delivery on microbial development and intestinal structure and function. Neonatal piglets were delivered either vaginally (VD) or by cesarean (CD) sow-reared for 21 days and then moved to cages and fed a weaning diet until d28. Samples were collected at d3, d14, d21 and d28 to examine effects of time. Mode of delivery did not affect digestive enzyme activity. Lactase and sucrase enzyme activities were measured. Lactase activity was variable over time (P<0.05), and sucrase activity increased over time (p<0.05). Both of these results were not unusual and reflect normal development in the piglet. Intestinal permeability was measured using the Ussing chamber. Route of delivery affect some of these parameters. CD piglets had greater glucose and glutamine transport at d14 compared to VD piglets. VD piglets also had increased jejunal basal Isc compared to CD piglets at all time points. In both the ileum and duodenum, 28-d-old piglets showed significant electrophysiological changes when compared to other time points, which s may be related to weaning. Resistance was increased at d28 in the duodenum compared to all other days, signaling that tight junction permeability was decreased. Intestinal histomorphology, villus height and crypt depth were evaluated and both day and mode of delivery had an effect, with VD piglets having longer villi than CD piglets and villi being longer at d14 than any other time point. Although, there were no significant differences in weight gain over time between the two groups, a slight separation in growth started to occur at d14 with the CD piglets slowing their rate of weight gain compared to VD piglets. These two results, in addition to the increased glucose and glutamine transport at d14, may be connected. Bacterial diversity was evaluated using Terminal Restriction Fragment Length Polymorphism (T-RFLP). There was no effect of route on this parameter. However, day did have an effect with the colonic communities separating out by day. Bacterial densities in the ileum and AC were quantified using qPCR and both universal and group specific primers. In the ileum, densities of total bacteria, Clostridium cluster XIVa and Lactobacillus were stable between d3 and 21, then decreased at d28. The numbers of ileal Bifidobacterium and Bacteroides-Prevotella did not change over time. In the AC, populations of total bacteria and Clostridium cluster XIVa increased from d3 till 14, were stable until d21, then decreased at d28. The numbers of Bifidobacterium and Lactobacillus were stable prior to weaning, and then increased and decreased, respectively, at d28. The densities of Bacteroides-Prevotella increased from d3 to d14 and were stable until d28. The products of bacteria fermentation, short chain fatty acids (SCFA), were affected by day, but not by route of delivery. Ileal butyrate concentrations decreased over time, reaching its lowest point at d28. Ileal acetate and propionate concentrations varied over time. In the ascending colon (AC), concentrations of all three short chain fatty acids (SCFA) remained stable through d21 and then increased at d28. Concentrations of branch chain fatty acids (BCFA) were only different in the AC, with isobutyrate and isovalerate being greatest at d14 and lowest at d28. Valerate concentrations in the AC were lower at d3 compared to all other days. In summary, changes in both intestinal parameters as well as microbiota were observed over time, many related to changes in diet at weaning. Route of delivery did not have the marked affects as previously hypothesized. This could be related to all the piglets being sow reared which may have lessened the impact of route of delivery on microbial colonization and in turn the other parameters of intestinal structure, function and permeability. The second aim of this dissertation focused on the effect of route of delivery and diet on intestinal function and immunity. Piglets were born either vaginally or through cesarean section and were fed one of three diets: formula, formula+prebiotic, or sow milk. The prebiotics chosen were polydextrose and scFOS (2g/L of each). The study duration was 14 days with a collection time point at d7 and d14. Dissacharidase and peptidase enzymes were examined in jejunal and ileal tissues. In the jejunum, neither day nor diet had an effect on lactase activity, but day had an effect on sucrase activity with d14 piglets having higher sucrase activity than d7. In the ileum, both day (p<0.0001) and diet (p<0.0001) had an effect on lactase activity with formula fed (FF) piglets having highest lactase activity followed by formula+prebiotic (FP) piglets, and then sow reared (SR). Lactase activity was also higher at d14 than d7. Ileal sucrase activity was also impacted by both day (p<0.0001) and diet (p<0.0001) with FF piglets having higher sucrase activity than both FP and SR piglets and activity was higher at d14 compared to d7. Aminopeptidase N (APN) was measured in both jejunum and ileum, and Dipeptidylpeptidase IV (DPPIV) was measured in jejunum and ileum, as well as serum. In the jejunum, both day (p=0.0023) and diet (p=0.0003) were significant, with d7 piglets having higher APN activity than d14. Both FF and FP piglets had higher APN activity compared to SR. In ileum, APN also had both day (p=0.047) and diet (p<0.0001) effects with FF piglets having significantly higher activity than FP and SR. Jejunal DPPIV showed effects of diet (p<0.0001) with FF piglets having higher activity than FP and SR. In ileum, both diet (p<0.0001) and day (p=0.0182) had effects with FF piglets having higher activity than FP and SR. There were no significant effects in blood. Ileal sections were stained for CD3+ T-lymphocyte populations. There was an effect of diet (p=0.0085) and day (p<0.0001) on population of CD3+ cells in the ileum. All groups had significantly more CD3+ cells at d14 compared to d7 and SR had significantly more cells compared to FF with FP being intermediate. Ileal cytokine expression was measured to evaluate Th1, Th2 and Treg cytokine expression. There were no differences between diet or day for this parameter. In summary, route of deliver did not affect intestinal structure or function, cytokine secretion or CD3+ cell populations in the intestine. The prebiotics, however, did show some effects with peptidase activity in both the jejunum and ileum as well as with CD3+ cell staining in the ileum. Formula+prebiotic piglets had more similar overall intestinal peptidase activity to SR piglets compared to FF. The addition of the prebiotic may have helped to modulate this result. However, FP piglets had significantly lower CD3+ cell expression in the ileum compared to SR. While the prebiotic creates similar effects to breast milk in some arenas, it still is not a complete replacement. The final aim of this dissertation focused on the effects of combined feeding and prebiotics on immune response and the colonic transcriptome. We developed a novel piglet model to investigate these effects. Newborn piglets were randomized into 5 groups: sow-reared (SR), formula fed (FF), formula+prebiotic (FP), combined fed (CF), and combined fed +prebiotic (CP) (n=6 per group). SR remained with the sow 24h/day and FF/FP were fed a sow milk replacer with or without prebiotics (GOS and Inulin; 2g/L each). CF/CP piglets were sow-reared for 5d and were then rotated between the sow and respective formula feeding group every 12h. On d21, piglets were intraperitoneally injected with 10ug/kg body weight of LPS. Four hours after infection, blood, mesenteric lymph node (MLN), spleen (SPL), and AC were collected and analysis performed. Immunity was tested by investigating both immune cell profiles and macrophage activation. T-cells, B-cells, dendritic cells, and macrophages were isolated, stained, and measured using a flow cytometer. In blood, SR showed significantly (p<0.0001) lower percentage of CD4+CD8- T-helper cells, while in the MLN they were intermediate between CP (p=0.0264) and all other diet groups. However, SR had a significantly (p<0.0001) higher percentage of B-cells compared to all other diet groups in blood. There were no significant differences between diet groups with dendritic cell or macrophage populations. Both acute phase response pro-inflammatory cytokines, IL-6 and TNF-α, were measured using ELISA. FF piglets had a much higher IL-6 response than both CF and SR diet groups. There was no significant difference in immune reaction to TNF-α. In addition, macrophage activation pathways, classical versus alternative, were also tested in both MLN and SPL tissue. Both nitric oxide synthase (classical) and arginase (alternative) enzyme activities were evaluated. There were no significant differences between diet groups when measuring nitric oxide synthase activity, but CP group did have significantly (p<0.0001) higher expression of arginase in MLN compared to all other diet groups. Combined feeding with prebiotic did have some effect in response to immune stimulus and seemed to encourage an anti-inflammatory immune profile as evidenced by the significantly higher arginase production which is caused by secretion of anti-inflammatory cytokines: IL-4, IL-10, IL-13 (Dewals et al, 2010). Gene expression of FF, CF, and SR piglets was assessed using a porcine gene microarray (Agilent). Among the 25,273 probes on the microarray, 449 were differentially expressed (FDR p<0.2). Bioinformatic analyses using MetaCore, revealed that the most significant GeneGo pathway maps were related to apoptosis, carbohydrate metabolism, lipid metabolism and neuronal pathway synthesis. Based on the pathways generated and the corresponding heatmap, CF and SR may have more similar gene expression patterns with genes related to lipid and carbohydrate metabolism, and apoptosis. FF and CF may have more similar gene expression patterns with genes related to glucose transport. More research is needed in this area to see how these differences affect future health outcomes

Limited effects of preterm birth and the first enteral nutrition on cerebellum morphology and gene expression in piglets

Preterm pigs show many signs of immaturity that are characteristic of preterm infants. In preterm infants, the cerebellum grows particularly rapid and hypoplasia and cellular lesions are associated with motor dysfunction and cognitive deficits. We hypothesized that functional brain delays observed in preterm pigs would be paralleled by both structural and molecular differences in the cerebellum relative to term born piglets. Cerebella were collected from term (n=56) and preterm (90% gestation, n=112) pigs at 0, 5, and 26days after birth for stereological volume estimations, large-scale qPCR gene expression analyses (selected neurodevelopmental genes) and western blot protein expression analysis (Sonic Hedgehog pathway). Memory and learning was tested using a T-maze, documenting that preterm pigs showed delayed learning. Preterm pigs also showed reduced volume of both white and gray matter at all three ages but the proportion of white matter increased postnatally, relative to term pigs. Early initiation of enteral nutrition had limited structural or molecular effects. The Sonic Hedgehog pathway was unaffected by preterm birth. Few differences in expression of the selected genes were found, except consistently higher mRNA levels of Midkine, p75, and Neurotrophic factor 3 in the preterm cerebellum postnatally, probably reflecting an adaptive response to preterm birth. Pig cerebellar development appears more affected by postconceptional age than by environmental factors at birth or postnatally. Compensatory mechanisms following preterm birth may include faster white matter growth and increased expression of selected genes for neurotrophic factors and regulation of angiogenesis. While the pig cerebellum is immature in 90% gestation preterm pigs, it appears relatively mature and resilient toward environmental factor

Mass spectrometric analysis of electrophoretically separated allergens and proteases in grass pollen diffusates

BACKGROUND: Pollens are important triggers for allergic asthma and seasonal rhinitis, and proteases released by major allergenic pollens can injure airway epithelial cells in vitro. Disruption of mucosal epithelial integrity by proteases released by inhaled pollens could promote allergic sensitisation. METHODS: Pollen diffusates from Kentucky blue grass (Poa pratensis), rye grass (Lolium perenne) and Bermuda grass (Cynodon dactylon) were assessed for peptidase activity using a fluorogenic substrate, as well as by gelatin zymography. Following one- or two-dimensional gel electrophoresis, Coomassie-stained individual bands/spots were excised, subjected to tryptic digestion and analysed by mass spectrometry, either MALDI reflectron TOF or microcapillary liquid chromatography MS-MS. Database searches were used to identify allergens and other plant proteins in pollen diffusates. RESULTS: All pollen diffusates tested exhibited peptidase activity. Gelatin zymography revealed high M(r )proteolytic activity at ~ 95,000 in all diffusates and additional proteolytic bands in rye and Bermuda grass diffusates, which appeared to be serine proteases on the basis of inhibition studies. A proteolytic band at M(r )~ 35,000 in Bermuda grass diffusate, which corresponded to an intense band detected by Western blotting using a monoclonal antibody to the timothy grass (Phleum pratense) group 1 allergen Phl p 1, was identified by mass spectrometric analysis as the group 1 allergen Cyn d 1. Two-dimensional analysis similarly demonstrated proteolytic activity corresponding to protein spots identified as Cyn d 1. CONCLUSION: One- and two-dimensional electrophoretic separation, combined with analysis by mass spectrometry, is useful for rapid determination of the identities of pollen proteins. A component of the proteolytic activity in Bermuda grass diffusate is likely to be related to the allergen Cyn d 1

Nutritional influences on human neurocognitive functioning

The notion that good nutrition is essential for adequate growth and sound physical wellbeing is very well established. Further, in recent years, there has been an overwhelming increase in research dedicated to better understanding how nutritional factors influence cognition and behavior (Riby et al., 2012). An aim of this Research Topic was to bring together Review, Opinion and Original Research articles reflecting the current science in this discipline. These include the effects of a range of foods and nutritional substrates on acute and chronic human neurocognitive functioning. The 13 accepted papers which form this Research Topic cover a diverse range of topics relating nutritional factors to neurocognitive functioning and performance. The articles demonstrate that neurocognitive performance is influenced by nutritional factors ranging from the dietary level (e.g., whole diet and meal composition) through to effects of macronutrients (such as glucose and omega-3 fatty acids) and micronutrients (vitamins, iron) on neurocognitive performance

In-pit crushing and conveying as an alternative to an all truck system in open pit mines

The material transport system in an open pit mine significantly affects the capital and operating costs. All truck haulage is the most common and is a reliable and flexible transport system. On the other hand, this system is very expensive and can cost up to 50% of total mining costs. Its cost is continuously increasing due to the inflation of the fuel, tire, and labour expenditures. In-pit crushing and conveying is an alternative transport system which requires a higher initial investment but gives substantial savings in operating costs. An evaluation of the all truck system versus the in-pit crushing and conveying system has been performed by means of a simulation of both transport systems in the same mine model. Results of the simulation and the data obtained from the feasibility studies provided input for an economic comparison of the alternative transport systems. A cash flow analysis showed that the in-pit crushing and conveying system was competitive with the all truck system, giving a payback within four years and resulting in total costs over 30% lower than those of an all truck system. Three computer programs, written by the author, have been used to analyse the mine model: (1) Open Pit Simulation Program - to model a hypothetical mine and simulate its haulage operation over the mine life, (2) Off-Highway Truck Simulation Program - to simulate the truck haulage on average annual routes in terms of the operating time and fuel consumption for the estimation of the truck fleet size and the fuel cost, (3) Cash Flow Analysis Calculation Program - to compare costs of the alternative transport systems over the whole period of a mine life.Applied Science, Faculty ofMining Engineering, Keevil Institute ofGraduat

Final Portfolio

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