Browsing by Author "Weir, Tiffany L., committee member"
Now showing 1 - 12 of 12
Results Per Page
Sort Options
Item Embargo Cardiovascular-protective effects of blueberry consumption in postmenopausal women with above-normal blood pressure(Colorado State University. Libraries, 2023) Woolf, Emily K., author; Johnson, Sarah A., advisor; Gentile, Christopher L., committee member; Weir, Tiffany L., committee member; Rao, Sangeeta, committee memberEndothelial dysfunction is the first step in atherosclerosis and contributes to its progression, and thus, is central to cardiovascular disease (CVD). It is driven by excessive oxidative stress and inflammation and characterized by impaired endothelium-dependent dilation. Estrogen-deficient postmenopausal women have oxidative stress-mediated suppression of endothelial function that is worsened by high blood pressure. Chronic blueberry consumption may be a beneficial dietary intervention for this population as it has shown to improve vascular function and blood pressure, though some studies have not demonstrated efficacy possibly due to the observed high interindividual variability in response to the intervention. Evidence indicates blueberries improve endothelial function, but studies have not been performed in postmenopausal women. Furthermore, ex vivo research has shown that blueberry (poly)phenols and their metabolites can decrease endothelial oxidative stress and inflammation, but whether these mechanisms translate to humans is unclear. The objectives of this dissertation were to 1) examine the efficacy of chronic blueberry consumption to improve endothelial function and blood pressure in estrogen-deficient postmenopausal women with above-normal blood pressure, with a specific focus on identifying mechanisms for improving endothelial function, 2) identify factors that contributed to the efficacy of blueberries as a dietary intervention for improving endothelial function, and 3) explore cellular mechanisms responsible for endothelial function improvements and the anti-atherogenic potential of blueberries. To investigate the aforementioned, we conducted a randomized, double-blind, placebo-controlled clinical trial and assessed endothelial function (measured through flow-mediated dilation (FMD)) and supine brachial blood pressure before and after daily consumption of 22 g of freeze-dried highbush blueberry powder or isocaloric placebo powder for 12 weeks. To examine mechanisms for improved endothelial function, FMD was assessed before and after infusing a supraphysiological dose of the antioxidant ascorbic acid (i.e. vitamin C) and normalized to shear rate area under the curve (FMD/SRAUC). To investigate factors impacting the interindividual variability in the endothelial function responses after the 12 weeks of blueberry consumption, we grouped the blueberry treatment group into responders (≥ +1% unit Δ FMD) and non-responders (< +1% unit Δ FMD) and performed secondary statistical analyses using data produced from the clinical trial. Lastly, to investigate mechanisms for improvements in endothelial function, we used a reverse translational human-to-cell approach leveraging human blood serum collected from participants in the clinical trial to perform ex vivo cell culture experiments. Results from the clinical trial showed that daily blueberry consumption significantly improved FMD/SRAUC compared to baseline by 96%. FMD not normalized for shear rate increased by 1.34% though the effects were not statistically significant (but were clinically significant). Improvements in FMD/SRAUC after blueberry consumption were due to reductions in oxidative stress as responses to ascorbic acid infusion were significantly reduced at 12 weeks in the blueberry group compared to baseline, with no changes in the placebo group. There were no major effects on blood pressure, arterial stiffness, endothelial cell protein expression, or other blood biomarkers of cardiovascular health. It was determined that the blueberry intervention was ~50% effective for improving FMD to clinically relevant levels of ≥ +1%, and that responders had decreased cardiovascular health and higher levels of circulating estrogen at baseline compared to non-responders. After 12 weeks of blueberry consumption, responders had reductions in oxidative stress, lower plasma nitrate levels, and higher phosphorylated endothelial nitric oxide synthase protein expression compared to non-responders. Lastly, we cultured HAECs with 15% serum (blueberry and placebo) for 1 h followed by 200 µM hydrogen peroxide (H2O2) for 24 h to induce endothelial dysfunction and evaluated the effects of blueberry (poly)phenol-rich serum on endothelial cell dysfunction and atherosclerosis progression. There were no statistically significant differences on monocyte binding, insulin-stimulated nitric oxide production, or peroxynitrite concentrations between dysfunctional HAECs treated with blueberry and placebo serum from the clinical trial. Collectively, results from these studies indicate that daily blueberry consumption for 12 weeks improves endothelial function in postmenopausal women with above-normal blood pressure through reductions in oxidative stress, and that efficacy (i.e. degree to which postmenopausal women responded to treatment in endothelial function) seems to be dependent on participant characteristics including cardiovascular risk factors and estradiol at baseline. Due to the inconclusive results regarding the ex vivo experiment, cellular mechanisms by which blueberry (poly)phenol metabolites impact endothelial function and atherosclerosis progression cannot be determined.Item Open Access Effects of various processing techniques and interventions on beef safety and shelf-life(Colorado State University. Libraries, 2017) Woerner, Christy M., author; Belk, Keith E., advisor; Martin, Jennifer N., advisor; Delmore, Robert J., committee member; Weir, Tiffany L., committee memberTwo experiments were conducted; the first evaluated decrease in log survival of pathogenic bacterial populations using three antimicrobial interventions (Peroxyacetic acid – PAA; Lactic Acid – LA; lactic/citric acid blend – LCA) applied at a spray cabinet used just before carcass chilling. Efficacy was evaluated using a Shiga-toxin producing Eschericia coli (STEC) inoculation cocktail that incorporated two strains of E. coli O157:H7 and 12 non-O157 STEC strains. In addition, this study was intended to validate the use of non pathogenic E. coli to serve as surrogates for the aforementioned STEC cocktail in plant operations. Influence of the carcass interventions on color stability of beef subprimals over a 30-day storage period was included to simulate effects on storage and display life. Each day, for three sampling days, 90 hot tissue samples from the plate subprimal were obtained immediately following slaughter. The tissue samples were evenly split into two inoculation groups (n = 45 samples/group): 1) STEC, or 2) surrogate. Within each inoculation group, samples were assigned randomly to one of nine treatments: i) 200 ppm PAA; ii) 1% LCA; iii) 1.5% LCA; iv) 2.5% LCA; v) 5% LA; vi) 8% LA; vii) 10% LA; viii) potable water; or ix) untreated control. Samples assigned to the surrogate inoculation group were further portioned into two equal sections for evaluation of the treatment influence on microbiological decrease in log survival and color. Samples were subjected to treatment using a custom-built, laboratory-scale spray cabinet to apply the intervention. Lightness (L*), redness (a*), and yellowness (b*) was evaluated before and immediately following spray application using a portable spectrophotometer. Following assessment of color immediately post-treatment application, the sample was further divided into three subsections that were vacuum packaged and stored for color evaluation at 10, 20, and 30 d. Among samples inoculated with STEC, log survival means with potable water and control were greater (P < 0.05) when compared to all other spray treatment groups. Likewise, the lower (P < 0.05) log survival means were observed for 8 and 10% LA treatment groups. No differences (P > 0.05) were observed among PAA, 1.5 and 2.5% LCA. Pairwise comparisons of surviving populations of STEC and surrogates revealed that the non-pathogenic strains could be effectively used as surrogates for the STEC cocktail. Color measures of L* values for samples spray treated with 8 or 10% LA were lower (P < 0.05) than for all other treatments, and declined over the 30 d storage period—indicating that the product darkened due to LA exposure and dark storage. Following 10 d dark storage, a* values were greater (P < 0.05) for untreated control samples than for samples sprayed with 1.5 or 2.5% LCA or for samples treated with any level of LA. Spectrophotometric b* values increased during dark storage (P < 0.05) suggesting product discoloration; however, no noticeable trends were observed among or between treatments. The second experiment monitored spoilage microorganisms, panelist and instrument color, and lipid oxidation changes during retail case display for three ground beef batches individually. After 7, 14, 18 or 21 d of vacuum-sealed, dark refrigerated storage (4C), three 73/27 ground beef batches (conventional - control; 25% inclusion of advanced meat recovery (AMR) product from plant one – BBFT 1; 25% inclusion of AMR product from plant two – BBFT 2) were separately fine ground, portioned into 454g loaves, and overwrapped with polyvinyl chloride (PVC) film for retail case display (4C) for 72 h. Sampling for aerobic plate count (APC), lactic acid bacteria (LAB) and 2-thiobarbituric acid reactive substances (TBAR) assay occurred every 24 h during retail case storage. Trained panelist-determined lean color, discoloration and redness intensity values, along with instrument L* (lightness), a* (redness) and b*(yellowness) measurements occurred every 12 h during retail case display. For each of the three products, neither least squares means for APC nor LAB exceeded 7 log CFU/g until after 21 d dark storage. Throughout retail case display, for all products, following all dark storage times, tan/brown discoloration means remained below 3%. With some exceptions, least squares means for panelist-determined lean color and redness intensity declined (P < 0.05) predictably, with greater retail case storage time. The L* means increased inconsistently depending on product or dark storage time; however, in several instances, L* values were highest (P < 0.05) toward the end of retail case storage. Conversely, a* values generally declined (P < 0.05) with increased storage times indicating a shift from bright red to dull blue color. Few noticeable trends among product and dark storage time were observed for CIE b* values throughout display. Least squares means for TBAR analyses were either similar (P > 0.05) or increased (P < 0.05) with retail case storage time.Item Open Access Efficacy of antimicrobial treatments against Salmonella enterica on pork and Campylobacter jejuni on poultry(Colorado State University. Libraries, 2020) González Sánchez, Sara Victoria, author; Belk, Keith E., advisor; Geornaras, Ifigenia, advisor; Delmore, Robert J., committee member; Weir, Tiffany L., committee memberTwo studies were conducted to evaluate efficacy of antimicrobial treatments against Salmonella enterica on pork and Campylobacter jejuni on poultry. The first study was conducted to (i) evaluate decontamination efficacy of six chemical treatments when applied to pork jowls inoculated with Salmonella enterica and (ii) determine the antimicrobial efficacy of the test solutions against a high and low inoculum level of Salmonella. Chilled pork jowls were cut into 10 × 5 × 1 cm portions and were surface-inoculated on the skin side with a mixture of six S. enterica serotype strains of swine origin. The inoculation levels targeted were 6 to 7 log CFU/cm2 (high) and 3 to 4 log CFU/cm2 (low). Following inoculation, samples were left untreated (control) or were treated by spray application (10 s, 18 to 19 psi, 1.0 gpm flow rate) with water, a proprietary blend of sulfuric acid and sodium sulfate (SSS, pH 1.2), formic acid (1.5%), peroxyacetic acid (PAA, 400 ppm), PAA (400 ppm) acidified with acetic acid (1.5%), PAA (400 ppm) acidified with formic acid (1.5%), or PAA (400 ppm) acidified with SSS (pH 1.2). Samples were analyzed for inoculated Salmonella counts immediately after treatment application (0 h) and after 24 h of refrigerated (4°C) storage. Overall, all seven spray treatments were effective (P < 0.05) at reducing the high and low Salmonella inoculation levels. At the high inoculum level (6.2 log CFU/cm2), pathogen counts ranged from 5.4 (water; 0.8 log CFU/cm2 reduction) to 4.3 (PAA acidified with SSS; 1.9 log CFU/cm2 reduction) log CFU/cm2 for samples analyzed immediately after spray treatment. Salmonella counts obtained at the 0-h sampling time for treated samples inoculated at the low inoculum level (3.5 log CFU/cm2) ranged from 2.8 (water; 0.7 log CFU/cm2 reduction) to 1.8 (PAA acidified with SSS; 1.7 log CFU/cm2 reduction) log CFU/cm2. Thus, regardless of inoculum concentration, similar reductions of Salmonella populations were obtained immediately following treatment application (0 h). For the high inoculation level, Salmonella counts of samples analyzed after 24 h of refrigerated storage were, in general, similar (P ≥ 0.05) to the counts of the corresponding treatment at 0 h. However, for the low inoculation level, pathogen counts of jowls treated with SSS, formic acid, or PAA acidified with formic acid, and held at 4°C for 24 h, were 0.6 log CFU/cm2 lower (P < 0.05) than the 0-h counts of the corresponding treatment. Regardless of inoculation level and sampling time, no (P ≥ 0.05) differences in efficacy were obtained between PAA on its own and any of the acidified PAA treatments evaluated. The second study was conducted to (i) evaluate decontamination efficacy of five chemical treatments when applied to chicken wings inoculated with Campylobacter jejuni and (ii) determine antimicrobial efficacy of the treatments as a result of applying test solutions by immersion or spraying. Skin-on chicken wings were surface-inoculated with a six-strain mixture of C. jejuni of poultry origin. The target inoculation level was 3 to 4 log CFU/mL of wing rinsate. Following inoculation, samples were left untreated (control) or were treated by immersion (500 mL solution per wing; 5 s) or spray application (10 to 12 psi; 4 s) with water, SSS (pH 1.2), formic acid (1.5%), PAA (550 ppm), PAA (550 ppm) acidified with SSS (pH 1.2), or PAA (550 ppm) acidified with formic acid (1.5%). Samples were analyzed for C. jejuni counts immediately after treatment application (0 h) and following 24 h of storage (4°C). All five acid treatments evaluated in this study were effective (P < 0.05) at reducing the initial inoculated (3.9 log CFU/mL) C. jejuni populations on chicken wings, regardless of the antimicrobial treatment application method. Pathogen counts for samples spray-treated with one of the chemical solutions and analyzed immediately (0 h) after treatment ranged from 3.4 (SSS; 0.5 log CFU/mL reduction) to 2.7 (PAA acidified with formic acid; 1.2 log CFU/mL reduction) log CFU/mL. When the chemical treatments were applied by immersion, C. jejuni counts of 2.2 (SSS; 1.7 log CFU/mL reduction) to 1.7 (PAA, and PAA acidified with SSS; 2.2 log CFU/mL reduction) log CFU/mL were obtained for wings analyzed at the 0-h sampling time. The PAA and acidified PAA treatments were equally (P ≥ 0.05) effective at reducing initial C. jejuni populations, regardless of treatment application method. However, following refrigerated storage, samples treated with SSS- or formic acid-acidified PAA had lower (P < 0.05) pathogen counts than those that had been treated with the non-acidified PAA treatment. Overall, findings of the two studies should be useful to the pork and poultry industries as they consider new interventions against Salmonella and Campylobacter contamination on pork and chicken parts, respectively.Item Open Access Elucidating rhizobacterial response to autoclave disruption and crop introduction within three distinct agricultural soils(Colorado State University. Libraries, 2020) DiLegge, Michael J., author; Vivanco, Jorge M., advisor; Manter, Daniel K., committee member; Weir, Tiffany L., committee member; Minas, Ioannis S., committee memberManagement practices can affect the soil health properties of an agroecosystem, in turn effecting the resident soil microbial community. Insights toward how managerial practices effect soil microbial rearrangements are steadily being uncovered with next generation sequencing applications. This thesis covers research investigating how soilborne and plant-rhizospheric bacteria from three differential agricultural management systems are affected by applied disruption followed by the introduction of new plants to their sites. Two independent greenhouse experiments were conducted to evaluate plant-mediated bacterial rearrangements in soil following autoclave disruption. The first study utilized two soil types from a perennial peach orchard system experiencing negative effects of orchard replanting disease. Soils were sampled from a replanting disease (RD) site and a non-replanting disease (non-RD) block. Replanting disease soils were autoclaved; and peach, corn and tomato plants were grown in both autoclaved and unautoclaved RD soils, as well as non-RD soils. Bacterial phyla and their predicted functional genomics were assessed after autoclave disruption and plant growth. The second experiment was an expansion of the former, utilizing autoclave disruption and the same perennial RD soil from the former study, but with the addition of conventional and organic annual agroecosystem soils. In this experiment four crops of differing plant families (corn, beet, tomato and lettuce) were introduced to examine how soil bacterial rearrangements may be influenced by distinct crop-presence after autoclave disruption. Results showed that autoclave disruption increased plant biomass. Interestingly, the type of crop plant introduced as well as the agroecosystem soil type drove differential bacterial responses and rearrangements. These data demonstrate that both agricultural ecosystem management, paired with the family of plants grown in these ecosystems, strongly impact soil bacterial availability and rearrangement in the rhizosphere. Additionally, in agricultural sites experiencing severe long-term dysbiosis, an autoclave disruption in pair with the rotation of monocultured crops may prompt the colonization of a healthier rhizomicrobiome.Item Open Access Elucidating the mechanisms of vascular dysfunction in obesity and type 2 diabetes: the role of the gut microbiota(Colorado State University. Libraries, 2019) Lee, Dustin Michael, author; Gentile, Christopher L., advisor; Weir, Tiffany L., committee member; Johnson, Sarah A., committee member; Chicco, Adam J., committee memberOne of the key processes that links both obesity and type 2 diabetes (T2D) to cardiovascular disease (CVD) is the development of vascular dysfunction, characterized by arterial stiffness and endothelial dysfunction. Vascular dysfunction occurs prior to overt CVD, and the development of vascular dysfunction in obesity and T2D strongly predicts future cardiovascular events and mortality. While the mechanisms of vascular dysfunction continue to be fully elucidated, an abundant body of research suggests that the gut microbiota mediate many cardiometabolic diseases. Disturbances to microbial equilibrium, broadly termed gut dysbiosis, have been implicated in numerous metabolic disorders. In a proof of concept study, our lab has previously demonstrated that suppression of gut dysbiosis reverses vascular dysfunction. Thus, further identifying useful and cost effective treatments that beneficially target the gut microbiota in obesity or T2D to prevent or reverse vascular dysfunction remains an important area of research. The goals of this dissertation research were to 1) examine the underlying causes of vascular dysfunction in models of obesity and T2D and 2) identify novel strategies to prevent or attenuate the development of vascular dysfunction in both obesity and T2D. To investigate the aforementioned, we conducted three separate preclinical studies utilizing a mouse model of T2D, diet-induced obesity, and gut microbiota transplantation. In these studies, we measured aortic pulse wave velocity and endothelium-dependent dilation to examine arterial stiffness and endothelial dysfunction, respectively. Both of these techniques are clinically relevant. We also employed several biochemical techniques to examine the mechanisms by which obesity and T2D lead to vascular dysfunction in our models. In the first study (Chapter 2), we explored epidemiological data suggesting that the antidiabetic drug class, sodium glucose cotransporter 2 inhibitors (SGLT2i), have beneficial effects on cardiovascular outcomes. Utilizing a genetic model of T2D to examine the vascular effects of SGLT2i, we found that treatment with dapagliflozin significantly improved both arterial stiffness and endothelial function. These changes were accompanied by decreased circulating inflammation and subtle alterations to the gut microbiota. In the second study (Chapter 3), we examined the effect of a gut microbiota-derived tryptophan metabolite on cardiometabolic outcomes. Mice fed a western diet displayed increased body weight, arterial stiffness and elevated markers of liver inflammation. Supplementation with the tryptophan metabolite, indole-3-propionic acid, had no effect on these outcomes. Finally, in the third study (Chapter 4) we examined whether human gut dysbiosis represents a causal factor in obesity-related vascular dysfunction. Utilizing human fecal samples from lean and obese subjects, we found that mice colonized with an obese gut microbiota displayed endothelial dysfunction independent of body weight changes. Collectively, these studies provide evidence that 1) SGLT2i-related cardiovascular protection is in part mediated by improvements in vascular dysfunction, 2) gut microbial metabolites have differing effects on host physiology, and 3) the obese human microbiota promotes endothelial dysfunction independent of body weight. Future studies should examine more mechanistic contributions of the gut microbiota that mediate vascular dysfunction in obesity and type 2 diabetes.Item Open Access Estimating potential biosafety risk of a CRISPR-Cas9 system for targeted killing of certain pathogens in beef cattle production using omic-based analysis methodologies(Colorado State University. Libraries, 2021) Dong, Jixin, author; Yang, Hua, advisor; Belk, Keith E., committee member; Metcalf, Jessica L., committee member; Weir, Tiffany L., committee memberThe CRISPR-Cas9 system has emerged as a programmable and versatile tool for precise gene editing purposes. In addition to gene editing, the CRISPR-Cas9 system can be developed to kill targeted bacteria. In our previous studies, we developed a CRISPR-Cas9 targeted killing system with a guide RNA designed to specifically recognize the Shiga-toxic genes (stx1 and stx2). Delivery of this system into E. coli cells could effectively kill Shiga-toxin producing E. coli (STEC) cells. This current study was conducted to estimate potential biosafety risk associated with our CRISPR-Cas9-based targeted killing system when applied to kill STEC cells in a bovine cell line model system using next generation sequencing (NGS) analysis. A bovine cell line CPA 47 (ATCC® CRL-1733™) was cultured to reach 90% confluence. Then the bovine cells were subjected to one of four treatments: 1) bovine cell control: without any CRISPR treatment; 2) CRISPR/gRNA: treated with phages that carry the CRISPR system with the guide RNA targeting stx genes (106 PFU/flask); 3) CRISPR+O157: treated with phages that carry the CRISPR system but without the guide RNA targeting stx genes (106 PFU/flask) and E. coli O157:H7 strain Sakai cells (105 CFU/flask); and 4) CRISPR/gRNA+O157: treated with phages that carry the CRISPR system with the guide RNA targeting stx genes (106 PFU/flask) and E. coli O157:H7 strain Sakai cells (105 CFU/flask). Each treatment was conducted in four replicates for a total of 16 samples. After application of treatments, bovine cells from each sample were collected and divided into two portions: half for whole genome sequencing (WGS) and half for protein analysis. Whole genome DNA from each sample was extracted, purified, and sent to Novogene Bioinformatics Technology (Beijing, China) for library construction and WGS. Raw reads were subjected to quality control (QC) procedures to remove unusable reads. Clean reads after QC were aligned to the bovine reference genome (NCBI access number: ARS-UCD 1.2 USDA ARS) using Burrows-Wheeler Aligner (BWA) with default parameter. Based on the mapping results, SAMtools was used to detect individual SNP/InDel variants, and ANNOVAR was used for functional annotation of the detected variants. A total of 1078 Gb of output with 3593.12 million paired end reads (150 bp) were obtained for all 16 samples after NGS. After QC, a total of 1073 Gb of clean data output were obtained for all 16 samples. The average output for the four replicates within the control, CRISPR/gRNA, CRISPR+O157, and CRISPR/gRNA+O157 treatments was 59.1, 72.3, 72.3, and 65.9 Gb, respectively. The mapping rate of each sample ranged from 99.48% to 99.75%, and the 4X coverage ranged from 89.45% to 98.23%. For SNP detection, a total of 94,796,157 SNPs were identified in all 16 samples when compared with the reference genome. The number of SNPs with each sample ranged from 5,225,269 to 6,192,930. Of the total number of SNPs, 60.01% were located in intergenic regions (regions between genes), 36.40% in intronic regions (non-coding sequences of genes), and 0.79% in exonic regions (coding sequences of genes). Further analysis of exonic regions showed that the average SNPs for each treatment of control, CRISPR/gRNA, CRISPR+O157, and CRISPR/gRNA+O157 was 0.1964%, 0.2002%, 0.2002%, and 0.1948%, respectively. SNPs within each functional class, for example, stop loss, stop gain, synonymous, non-synonymous, at slicing sites, and upstream or downstream from transcription termination sites, the number of SNPs all showed no significant differences (P > 0.05) among the control and the three CRISPR treatments. For InDel detection, a total of 11,949,421 InDels were identified in all 16 samples, with each sample having between 604,387 and 817,716 InDels. Of the total number of InDels, 62.78% were located in intergenic regions, 38.54% in intronic regions, and 0.15% in exonic regions. The average exonic InDels in the control, and CRISPR/gRNA, CRISPR+O157, and CRISPR/gRNA+O157 treatments was 0.0371%, 0.0372%, 0.0375%, and 0.0372%, respectively. InDels within each functional class, for example, stop loss, stop gain, frameshift insertion/deletion, non-frameshift insertion/deletion, at slicing sites, and upstream or downstream from transcription termination sites, the number of InDels all showed no significant differences (P > 0.05) among the control and the three CRISPR treatments. Neither the SNP nor InDel data showed significant differences (P > 0.05) in the number of SNPs/InDels between the control and the CRISPR treatments when their WGS data were compared with the bovine reference genome. These results go along with our initial prediction that the biosafety concern of our CRISPR-Cas9 system should be low because our CRISPR system was designed to make a cleavage on target bacterial genomes but not on cattle genomes. In addition to coding regions, we are continuing with our analysis of the variants in non-coding regions. Results from this study will provide insights on how to further improve approaches or develop criteria on biosafety evaluation of the CRISPR-Cas9 system. Completion of this project will provide the beef industry with biosafety information regarding application of CRISPR as an alternative to antibiotics in cattle production.Item Open Access Evaluation of GENE-UP and TEMPO AC for determination of Shiga-Toxin producing Escherichia coli and total aerobic microbial populations from MicroTally sheets used to sample beef carcasses and hides(Colorado State University. Libraries, 2020) Liu, Tianqing, author; Belk, Keith E., advisor; Yang, Hua, advisor; Weir, Tiffany L., committee member; Zagmutt, Francisco J., committee memberTwo studies were conducted to evaluate GENE-UP and TEMPO AC (bioMerieux, Marcy-l'Étoile, France) for determination of Shiga-Toxin producing Escherichia coli and total aerobic microbial populations from MicroTally Sheets (Fremonta Corporation, Fremont, CA) used to sample beef carcasses and hides. The first study was conducted to evaluate the automated TEMPO® AC Test in comparison with traditional direct agar plating method for enumeration of aerobic mesophilic flora in MicroTally sheets used to sample beef carcasses and hides. A total of 160 MicroTally (MT) sheet samples were collected from commercial beef processing plants by swab-sampling on the surface of naturally contaminated pre-evisceration carcasses, hides and post-chill final carcasses, and analyzed within 24 h after sample collection. Of these, all 160 samples were within detection limit and analyzed by both automated TEMPO AC test and a traditional direct agar plating method. For these results, the aerobic count correlation coefficient was high (0.93) for pre-evisceration carcasses, which had mean (± standard deviation) counts of 3.3 ± 0.9 and 3.1 ± 0.8 log CFU/mL for those two methods, respectively. The aerobic count correlation coefficients were higher (0.95 and 0.96) for MT samples from hides and post-chill final carcasses, which had mean (± standard deviation) counts of 5.3 ± 1.2 and 5.0 ± 1.2, 3.0 ± 1.4 and 3.0 ± 1.3 log CFU/mL for those two methods, respectively. Overall, 98.8% of aerobic count results were within 1.0-log difference between the two enumeration methods. The correlation coefficient (r = 0.97) and linearity regression (log TEMPO MPN/mL = 1.06 x log PCA-CFU/mL +0.03) between the two methods was calculated for our whole sample set (n = 160). Our results demonstrated that the automated MPN method-TEMPO AC Test generated total aerobic mesophilic microflora counts that were highly correlated and consistent with the counts obtained by traditional plating methods on enumerating total aerobic mesophilic microbial populations recovered from MicroTally sheets. Use of TEMPO AC test for MicroTally sheet analysis could save time and labor for the meat industry as it conducts microbial analyses. The second study was conducted to determine the specificity of bioMérieux's GENE-UP, a PCR-based molecular diagnostic system, to detect Shiga Toxin-producing Escherichia coli (STEC) from samples collected from beef processing plants using MicroTally sheets with the manual sampling device method. A total of 194 MicroTally (MT) samples were collected from beef processing plants and analyzed for determination of the top 6 STEC and E. coli O157: H7 (top 7 STEC) using the GENE-UP system, BioRad commercial kits and BioControl GDS kits. Fifty MT samples were collected from swabbing pre-evisceration carcasses and inoculated with hide-derived inocula, while the remaining 144 MT samples were obtained from post-chill final carcasses in sales coolers and inoculated with E. coli strains. All inoculated MT samples were enriched for 8-hour and 10-hour at 42ᵒC in buffered peptone water (BPW) and re-collected after incubation. Eight-hour and 10-hour enrichment samples were analyzed using the GENE-UP system at Colorado State University and sent to U.S Meat Animal Research Center (USMARC, Clay Center, NE) for detection of top 6 STEC and E. coli O157: H7. The GENE-UP system uses EH1 assay to detect stx and eae genes, ECO assay to detect genes specific to O157:H7 serogroup, and EH2 assay to differentiate top 6 serogroups. These virulence genes including Shiga-toxin gene (stx), intimin-encoding eae gene and genes specific to top 7 serogroups are highly related to pathogenic STEC. The NM-EHEC assay targeting virulence genes espK, espV and CRISPR_O26E does not directly differentiate the top 7 STEC, but serves as additional screening test to help identify presence of any of the top 7 STEC. All potential positive samples determined by PCR screening were plated onto selective agar for culture confirmation. After the immunoconcentration step, isolates picked from selective agar were subjected to additional PCR screening. BioRad and BioControl GDS PCR screening methods were used following their standard protocols for determination of top 7 STEC at USMARC. Presumptive positive samples confirmed by the additional PCR test were designated as "true positives." Presumptive positive samples that were not confirmed by the additional PCR test were designated as "regulatory false positives." Overall, our results indicated that the GENE-UP system worked well in the detection of the top 7 STEC recovered from the MicroTally sheets. In order to reduce or eliminate false negative results, a 10-h enrichment time in BPW was required for detection of both the top 6 STEC and E. coli O157:H7. Compared to GENE-UP and GDS, BioRad generated a much higher number of potential positives that required cultural confirmation. Moreover, use of the NM-EHEC kit targeting virulence genes (espK, espV and CRISPR_O26E), as an additional PCR screening after EH1 PCR (stx and eae), has potential to reduce the number of samples that require further O-type determination. However, the GENE-UP E. coli O157:H7 detection system needs to reduce rates of false negative results caused by the shift of Tm when E. coli O157:H7 and O157: non-H7 co-exist in a sample.Item Open Access Implications of diet-induced obesity on metabolic and immune homeostasis: the role of the mesenteric lymph nodes(Colorado State University. Libraries, 2020) Hill, Jessica Lynn, author; Michelle, Foster T., advisor; Weir, Tiffany L., committee member; Gentile, Christopher L., committee member; Schenkel, Alan, committee memberObesity is a major public health crisis among adolescents and adults. The development of obesity is associated with several comorbidities as a result of underlying systemic chronic inflammation, the culmination of which increases one’s risk for chronic and infectious disease. Excessive accumulation of visceral adipose tissue is shown to confer the greatest disease risk. This is primarily due to inherent depot differences, namely proximity to and a shared blood supply with the liver and gastrointestinal (GI) tract. Recent work demonstrates the considerable influence gut physiology has over both local and systemic homeostasis, as GI diseases such as inflammatory bowel disease are associated with metabolic derangements characteristic of obesity. While the mechanisms that mediate this inter-organ crosstalk continue to be elucidated, several studies suggest that inflammation originating from the gut triggers these broad metabolic and immunologic changes found in obesity. Previous work from our lab has demonstrated that high-fat diet (HFD) induced obesity results in mesenteric lymph node (MLN) fibrosis, which was associated with a localized impairment in immune function. MLNs, located within mesenteric adipose tissue (MAT) surrounding the GI tract, constitutively monitor the mesenteric adipose depot and draining sections of the small and large intestines, serving as critical inductive sites for adaptive immune responses. Subsequently, they are essential for overall tissue maintenance and protection. Hence, further study into the role of the MLNs in obesity-associated pathology is an important area of research. The goals of this dissertation research were to 1) examine the relationship between MLNs and GI inflammation on metabolic outcomes, and 2) characterize immunologic changes associated with models of chronic inflammation. To investigate the above-mentioned, we conducted four separate preclinical studies utilizing mouse models of diet-induced obesity, MLN cauterization, and dextran sulfate sodium (DSS) induced GI inflammation. In the first study (Chapter 2), we examined the contribution of the MLNs on disease pathology associated with HFD-induced obesity. We found that MLN dysfunction, either as a result of surgical manipulation or obesity-induced fibrosis, led to metabolic dysfunction. Furthermore, that functional MLNs are needed for the full restorative effects of Pirfenidone treatment. In the second study (Chapter 3), we examined the effect of chronic low-dose DSS induced GI inflammation, independent of diet and obesity, on metabolic and immune function. We found that non-obese mice treated with DSS had a modest reduction in total body weight and MAT mass yet showed substantial alterations in tissue immune cell populations and frequencies. These adaptations occurred without a concurrent change in glucose homeostasis. Finally, in the third study (Chapter 4) we characterized immunologic parameters within a normal weight and obese human population, free of disease, through the ex vivo challenge of peripheral blood mononuclear cells (PBMCs) with the T lymphocyte mitogen Concanavalin A (ConA). We found that PBMCs isolated from obese adults had a modest increase in cell proliferation and IFNγ secretion upon stimulation within ConA relative to their normal weight controls. Additionally, we found a distinct expansion of CD4+CD8+ T cells, CD16+ monocytes, and NK cells within ConA stimulated PBMCs from obese donors. Collectively, these studies provide evidence that 1) the MLNs are critical for metabolic homeostasis as their dysfunction exacerbates features of HFD-induced obesity; 2) chronic GI iv inflammation, independent of diet and obesity, can reshape the immune milieu without altering glucose homeostasis; and 3) obesity distinctly alters the PBMC response to acute ex vivo challenge as compared to that of normal weight individuals. Future studies should further elucidate mechanisms of crosstalk between the immune system, MLNs, and GI tract on metabolic homeostasis in models of obesity.Item Open Access Nutritional composition and food safety interventions of plant and animal-sourced foods(Colorado State University. Libraries, 2021) Swing, Caleb John, author; Nair, Mahesh Narayanan, advisor; Geornaras, Ifigenia, committee member; Weir, Tiffany L., committee member; Belk, Keith E., committee memberNutritional composition of plant- and animal-sourced food is important for human growth and development, and yet even nutritious food-groups can be detrimental to human health if contaminated with harmful pathogens upon consumption. Therefore, two studies were performed to assess the nutritive quality of plant- and animal-sourced proteins; as well as, the antimicrobial efficacy of novel sanitizers against a foodborne pathogen attributed to illness from plant- and animal-sourced food consumption. In the first study, nutrient profiles of animal-derived meat products, which are traditionally an important source of nutrients in the human diet, were compared to novel plant-based meat alternatives, which have been growing in popularity among modern consumers. Nutritional composition of two different formulations of the Beyond Meat Burger (BMB1 and BMB2), Impossible Food Burger (IFB1 and IFB2), 80/20 ground pork (GP), and 80/20 ground beef (GB) were analyzed for proximate, mineral, vitamin, fatty acid, and amino acid profiles. Crude protein and crude fat content did not differ (P > 0.05) for each product in cooked states. Plant-based meat alternatives were either numerically greater than or did not differ statistically (P < 0.05) from animal-derived meat products in every mineral tested. Fat soluble vitamin A, D2, D3, and K1 were below detection limits (< 0.3 mcg/g for vitamin A; < 0.001 mcg/g for vitamin A, D2, D3, and K1) in all raw and cooked samples. Vitamin E content in raw and cooked plant-based meat alternatives was substantially greater (P < 0.05) than in raw and cooked animal-derived meat products. Raw and cooked GP and GB were substantially greater (P < 0.05) than IFB1 and IFB2 in pantothenic acid (B5) but otherwise were numerically similar to or statistically less (P < 0.05) than IFB1 and IFB2 in most B vitamins tested. Total saturated and monounsaturated fatty acids did not differ (P > 0.05) for BMB2, IFB2, GP, and GB. IFB1 and IFB2 were greater (P < 0.05) than GP and GB in oleic acid (C18:1) content. Fatty acid profiles of raw and cooked BMB2 and IFB2 did not differ (P > 0.05) from one another. Essential amino acid composition of raw and cooked plant-based meat alternatives and animal-derived meat products were numerically comparable. Raw BMB2 did not differ (P < 0.05) from raw GP in histidine, lysine, and threonine content and was otherwise greater (P < 0.05) than raw GP in tyrosine, isoleucine, leucine, and valine. Raw GP was only numerically greater (P > 0.05) than raw BMB2 in methionine and tryptophan. In conclusion, plant-based meat alternatives assessed in this study were comparable to animal-derived GP and GB in most nutrient profiles assessed, providing high values of minerals, vitamins, fatty acids, and amino acids. Nonetheless, the high concentrations of certain nutrients as well as the integration of these nutrients into a food matrix may have implications for bioavailability and must be further investigated. In the second study, efficacy of novel antimicrobial sanitizers was assessed in relation to reducing Listeria monocytogenes contamination on a plant-based food. Both plant and animal-sourced foods have proven to be vectors of L. monocytogenes contamination, but a largescale, multistate listeriosis outbreak was attributed to whole cantaloupes raising concerns for the potential contamination of other fresh produce not previously associated with L. monocytogenes contamination. This study assessed efficacy of chlorine as well as different concentrations of novel sanitizer and sulfuric-acid based surfactant blends, peroxyacetic acid (PAA) and ProduceShield Plus (PSP), against inoculated L. monocytogenes populations on whole cantaloupe melons (Cucumis melo L. var. reticulatus). Cantaloupe melons (n = 6) were inoculated with a five strain mixture of L. monocytogenes (7 - 8 log CFU/cantaloupe) and immersed in water, chlorine (40 ppm), PSP (pH 1.81), PAA (40, 80, 250 ppm), or PAA+PSP (40, 80, 250 ppm and PSP blend) sanitizer solutions, under slight agitation for 0.5, 1, and 5 min exposure times. Recovery of surviving L. monocytogenes populations after immersion treatment, was accomplished by vigorously shaking whole cantaloupes in D/E neutralizing broth and plating the rinsates on PALCAM agar. The L. monocytogenes inoculation level achieved on whole cantaloupes was 7.9 ± 0.4 log CFU/cantaloupe. Immersion of inoculated whole cantaloupes in water or PSP achieved pathogen reductions that ranged between 0.3 to 0.5 log CFU/cantaloupe, and 0.9 to 1.8 log CFU/cantaloupe, respectively, across the three different exposure times (0.5, 1, 5 min). Reductions of L. monocytogenes populations on inoculated cantaloupes treated with 40 ppm chlorine achieved less than or equal to 3.3 log CFU/cantaloupe reductions across the different exposure times; while different concentrations of PAA (40, 80, 250 ppm) all achieved greater than or equal to 3.1 log CFU/cantaloupe reductions across the three exposure times. Different concentrations of PAA (40, 80, 250 ppm) blended with PSP resulted in pathogen reductions of between 3. 2 and > 4.9 log CFU/cantaloupe across the different exposure times. Decontamination efficacy of each PAA concentration level, within each treatment and exposure time, was similar (P > 0.05) to that of its corresponding PAA+PSP blend for most cases, although the PAA+PSP blends had numerically greater reductions than each corresponding PAA treatment and contained several samples which were below the detection limit of (2.7 log CFU/cantaloupe). In summary, PAA and the PAA+PSP blends demonstrated the greatest antimicrobial efficacy against L. monocytogenes populations on inoculated whole cantaloupes. More research should be conducted to elucidate a possible synergistic effect between PAA and sulfuric acid-based surfactants, such as PSP, on plant and animal-sourced foods susceptible to L. monocytogenes contamination.Item Open Access Potato yield and nutrient acquisition are supported by the soil "bacteriome"(Colorado State University. Libraries, 2013) Barnett, Brittany Allison, author; Manter, Daniel K., advisor; Bunning, Marisa L., committee member; Holm, David G., committee member; Vivanco, Jorge M., committee member; Weir, Tiffany L., committee memberPotatoes are the fourth largest food crop in the world; they are a staple food for much of South America and are the most consumed vegetable per capita in the United States. Breeding programs across the country seek to produce cultivars that are high yielding, disease resistant, and nutritious. The plant-soil-microbial community is greatly intertwined, each piece affecting the others. Soil microbial communities are highly influenced by edaphic features, and within a site microbial communities are influenced by the specific potato clone. The first section of this project (Chapter 2) illustrates the variability in the bacterial root-associated community due to site and clone. The underlying core bacterial community of combined potato roots/rhizosphere soil that might benefit the quality of the potato crop was also examined. Root/rhizosphere soils from 18 different clones along with bulk soil bacterial communities from three sites (CA, CO, TX) were examined using 454 sequencing. In order to explain the soil bacterial potential, SPLS regression techniques were used to identify root-associated microbes correlated with tuber yield. Twenty-two bacterial operational taxonomic units (OTUs) were found to have a significant positive relationship with potato yield, many of these belonging to the bacterial order Rhizobiales. Interestingly, many of the bacteria identified in the SPLS regression have been studied in agricultural systems, however rarely in relation to potato. Further study of the relationship between potato plants and these microbes is warranted. Parts of South America, where potato is a staple food, have been described as good candidates for the implementation of biofortified foods; additionally, the potato is a good candidate for biofortification. Biofortification of foods through plant breeding can increase essential nutrients in staple crops to decrease human disease and mortality. The second aspect of this project (Chapter 3) assessed the impact that soil nutrient contents, soil bacterial diversity, and potato clones have on tuber nutrient contents. A predictive model was created to address the degree to which these independent predictors impact the tuber nutrient levels of N, P, K, Zn, Fe, Mn and Cu. Soil nutrient levels and bulk soil bacterial diversity had a similar ability to increase tuber nutrient levels. Increasing soil bacterial diversity was shown to support acquisition of these seven nutrients. This indicates that management practices to increase soil bacterial diversity may support plant nutrient acquisition, thus lowering fertilizer use.Item Open Access Relationships between plasma cytokines, leukocyte telomere length, serum lipid profile, and nutrient intake in healthy adults following a 4-week dietary intervention study(Colorado State University. Libraries, 2016) Harbison, Gregory James, author; Ryan, Elizabeth P., advisor; Bailey, Susan M., committee member; Tjalkens, Ronald B., committee member; Weir, Tiffany L., committee memberColorectal cancer is the third most commonly diagnosed cancer worldwide and the fourth leading cause of cancer-related death. The etiology of colorectal cancer is predominately attributed to modifiable lifestyle factors that promote chronic inflammation, and only 20% of colorectal cases are credited to hereditary syndromes. Specifically, recent nutritional studies have suggested that diet modification is a promising lifestyle intervention for reducing systemic inflammation and promoting colorectal cancer prevention and remission. In particular, rice and navy beans have been identified as two foods with anti-inflammatory and anti-neoplastic properties that warrant evaluation for chemoprevention through dietary supplementation in humans. In this study, plasma cytokines (IL-2, IL-4, IL-6, IL-8, IL-10, TNF, and VEGF) and leukocyte telomere length were measured at baseline, two weeks, and four weeks in individuals with and without a history of colorectal cancer who consumed a diet supplemented with rice bran, navy beans, or a placebo-control for 28 days. Serum lipid profile and nutrient intake were also measured. At baseline, the three diet intervention groups had no significant differences in cytokine concentration, telomere length, or lipid profile. At the end of the study, individuals with a history of colorectal cancer who consumed the navy bean supplemented diet had significantly higher plasma TNF and VEGF concentrations than individuals consuming the control diet. Otherwise, at the end of the study, no significant differences in cytokine concentration or telomere length between groups existed. Additionally, compared to males, females with a history of colorectal cancer had significantly longer telomeres at baseline but not at four weeks. Females with a history of colorectal cancer also had significantly lower IL-4, IL-6, and IL-10 at baseline, but no significant difference was found at four weeks. Linear correlation analysis on repeated measures that adjusted for sex, age, and total energy intake showed significant correlations between several study variables. Telomere length was inversely correlated with age, serum triglyceride level, carbohydrate intake, and saturated fat intake. IL-2 and IL-4 concentrations were inversely correlated with α-Tocopherol intake. IL-8 was inversely correlated with vitamin B3 intake. VEGF was positively correlated with vitamin B9 intake. Total serum cholesterol was positively correlated with saturated fat intake and inversely correlated with β-Carotene intake. Serum LDL was inversely correlated with β-Carotene intake, and serum HDL was positively correlated with intake of saturated fat and linolenic acid. Triglyceride level was inversely correlated with intake of β-Carotene and fiber and was positively correlated with selenium intake. Finally, comparison of two experimental methods for telomere length measurement showed positive but inconclusive correlations.Item Open Access The survival of inoculated populations of Listeria monocytogenes and Staphylococcus aureus on shelf-stable meat bars during vacuum-packaged storage(Colorado State University. Libraries, 2018) Bullard, Brittney Rose, author; Delmore, Robert J., advisor; Belk, Keith E., committee member; Geornaras, Ifigenia, committee member; Martin, Jennifer N., committee member; Weir, Tiffany L., committee memberTo view the abstract, please see the full text of the document.