Progress 05/01/10 to 04/30/15
Outputs
Target Audience:Producers and distributors of pinto beans, most noteably those located within the central high plain states, i.e., NE, CO, and WY. Changes/Problems:The major problem is that funds were not available to work with the primary cell lines. Therefore, much of the research cited in the proposal was accomplished with immortal macrophages. However, as stated previously, due to the data obtained from this research, funds have been obtained to continue this project. What opportunities for training and professional development has the project provided?Three graduate students, 2 undergraduates, and a visiting scholar were trained on the methodologies required to characterize phenolics from dry beans, and/or intermediates of the global metabolism / central carbohydrate pathway in both macrophages and C. albicans. How have the results been disseminated to communities of interest?At this point, dissemination has been through student seminars, internal presentations, and newsletters targeted to pinto bean producers and distributors. In addition, mulitple papers for peer-reviewed journal are now in progress. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
A significant issue facing the United States dry edible bean industry is the current price of competing field crops, such as corn, and the influx of dry edible beans from international sources. Considering that Nebraska is the No. 3 producers of the pinto bean in the United States, many counties throughout our state have been affected by this decline particularly those in the Panhandle, i.e., an area already adversely affected by decreasing populations and low income growth. As the number of acres of dry edible bean production declines, so too does their economic viability, which, in turn, has disrupted the rest of the economies in this region. Therefore, the expected impacts of this research is that by understanding the antiinflammatory properties of pinto beans based upon their ability to modulate cellular metabolomes towards a healthy phenotype we will be better positioned to develop consistently safe and efficacious food systems derived from dry edible beans. This research thus supports Nebraska and US dry edible bean agriculture by promoting the consumption of pinto beans for improved and sustained human health. Specific Aim 1: The key outcome from this specific aim is that metabolomics methods and protocols were completed using a C. albicans model system, as described in previous reports. However, it should be noted that spin-off research has evolved from this work, which consists of developing multi-targeted antifungals using phenols abundant in beans and many natural product waste lines. Therefore, another key accomplishment from this preliminary research was that dietary agents, which have been linked to strengthen our immune system, were also able to remediate or prevent the virulence of an organism responsible for causing 30-40% rates of life-threatening infection. Our approach was unique in that dietary phenols present in dry edible beans (ferulic acid, gallic acid, and sinapic) were initially identified based on their ability to remediate / prevent the non-virulent C.albicans phenotype (yeast) from transitioning into the virulent state (hyphae). It was determined that ferulic acid, gallic acid, and sinapic acid, were able to act together to inhibit the hyphae form, but the effect is dosage and time dependent (data not shown). The second phase of these experiments was to prevent tissue invasion of the hyphal phenotype, another criterion for virulence, via dietary phenolic synergists. Therefore, the ability of several phenols were monitored to reduce reactive oxygen species (ROS) that builds in C. albicans as it transitions to the hyphal stage. (The eventual burst of ROS has been linked to tissue invasion.) Our studies showed that certain bean based flavonoids were not able to remediate the hyphal phenotype are able to significantly reduce the internal ROSs. FTIR and other metabolomics based data that has accumulated from the methods established from this specific aim are currently being used to determine the mechanisms of action of the phenols on C.albicans, particularly on how global metabolism (non-targeted), and central carbohydrate metabolism (targeted) are modulated by the phenols to maintain the non-virulent state. Moreover, these methods are now being used on immortal cell lines in response to isolated pinto based phenols as well as extracts of pinto beans. Specific Aim 2: The key outcome from this specific aim is that metabolomics methods and protocols were established that are able to detect subtle and temporal changes in the M1 and M2 phenotype of Raw 264.7 macrophages. These methods included capillary electrophoresis, (specifically for lactic acid, AMP/ATP, and NADH / NAD+) as these intermediates differ between the phenotypes, as M1 exhibits higher levels for other three parameters. Moreover, a FTIR method was also established to allow high throughput analysis between the M1 and M2 state. The data are currently being analyzed. Moreover, colorimetric assays were also set up to measure nitric oxide (NO), which is high in M1 macrophages, but low in M2. Lastly methods were established to determine TCA cycle, nucleotide, amino acid intermediates again using capillary electrophoresis, whereas ion exchange - amperometric chromatography was used to monitor intermediates of glycolysis and the pentose phosphate pathways. (The development of these methods and their application is documented in the dissertation of my former Ph.D. student, and a paper is in preparation.) Using these methods, the ability of phenols to modulate the M1 and M2 state are currently being studied. However, immortal macrophages were utilized instead of the originally planned bone marrow primary lines due to lack of resources to secure those cells. (However, we have recently secured funding to proceed in primary cell lines). Still, promising preliminary studies have been obtained from the Raw 264.7 macrophage line using phenols present in beans but as isolated components. First, concentrations were determined that did not affect the viability of the cells were first determined using the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The macrophages were then subjected to lipopolysaccharide, (LPS), i.e., inducer to the M1 state, for 24 hours, followed by treatment (1 ng or 10 ng) with different phenols. Nitric oxide and lactic acid was monitored to determine whether the phenols were able to remediate the bean M1 stateback to baseline or possible to the M2 state (data not shown). Nitric oxide was monitored for macrophage exposed to LPS only (inducer of M1), no LPS (inactivated) and Il4 (inducer of M2). Single doses of most of the phenols did not affect the M1 state, with the notable exception of 4-vinyl phenol (1 and 10 ng), catechins (10 ng), and sinapic acid (10 ng). Based upon this data, two phenols were combined and the cells were treated once again in a dose dependent manner (ranging from 0.5:0.5 ng per phenol to 5:5 ng per phenol) after 24 hours post induction of the M1 state with LPS. Combined, the phenols were able to reduce the M1 phenotype to baseline at much lower concentrations with 5 ng of kaempferol and 5 ng protocatechin remediating M1 to baseline (data not showne). Interesting, these two phenols were not able to substantially modulate the M1 used as isolated components at 10 ng each. Moreover, kaempferol-3-glycoside (a major flavonoid in pinto beans) was even able to modulate the M1 phenotype to a M2 state, for at least some of the cells, when combined with pyrogallol. However, 100% of the cells were modulated from the M1 state to the M2 state with 5 pyrogallol was combined wiht farnesol, i.e., a small molecule with not present in beans, but naturally occuring in other natural systems. Various extracts from pinto beans, (obtain from a RSM model) and characterized accordingly are showing similar responses. Isobolograms are also now being constructed to understand if the phenols act in synergism or as addivtives. Lastly, metabolomic data from the macrophages have also been collected and now being analyzed to understand how central metabolism was affected by the bean treatments. Specific Aim 3: Specific Aim was not started yet, due to inability to obtain resources to support the primary cell work. However, due to the data obtained above, funding has now been secured to continue this project with primary cell lines.
Publications
- Type:
Theses/Dissertations
Status:
Other
Year Published:
2015
Citation:
One Ph.D. student will graduate in Dec 2015 based on work performed under this project. The other two graduate students (Ph.D.) anticipated graduation dates are Spring 2016 and Dec 2016.
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience:
Nothing Reported
Changes/Problems: The major problem is that funds were not available to work with the primary cell lines. Therefore, much of the research cited in the proposal was accomplished with immortal macrophages. What opportunities for training and professional development has the project provided? Three graduate students are currently being trainied on the methodlogies required to characterize phenolic from dry beans, and intermeidates of the central carbohydrate pathway in both macrophages and C. albicans. All the students are Ph.D. candidates have presented their work on various internal retreats and seminars. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals? Items to complete are stated in the Accomplishment sections but include: Specific Aim 2: A.) Isobolomograms for syneristic phenols able to modulate the M1 to M2 state. B.) Complete other tests to confirm the M1 and M2 state such as cytokines, lactic acid, AMP/ATP. C.) Determine how the phenols act synergistically to modulate central carbohyrate metabolism using immortal cell lines. D.) test extract obtained from pinto beans and determine their ability to modulate the M1 to M2 phenotype. Specific Aim 3: A.) Based on the data and the most potent combination of pinto bean extracts identified in Specific Aim 2, determine their ability to modulate the M1 and M2 state using primary cell lines. B.) Identify the phenols responsible for this phenomenom and if they are acting as synergists, C.) Study how central carbohydrate metabolims is impacted by the efficacious extracts.
Impacts
What was accomplished under these goals?
A significant issue facing the United States dry edible bean industry is the current price of competing field crops, such as corn, and the influx of dry edible beans from international sources. Considering that Nebraska is the No. 3 producers of the pinto bean in the United States, many counties throughout our state have been affected by this decline particularly those in the Panhandle, i.e., an area already adversely affected by decreasing populations and low income growth. As the number of acres of dry edible bean production declines, so too does their economic viability, which, in turn, has disrupted the rest of the economies in this region. Therefore, the expected impacts of this research is that by understanding the anti-inflammatory properties of pinto beans based upon their ability to modulate cellular metabolomes towards a healthy phenotype we will be better positioned to develop consistently safe and efficacious food systems derived from dry edible beans. This research thus supports Nebraska and US dry edible bean agriculture by promoting the consumption of pinto beans for improved and sustained human health. Specific Aim 1: The key outcome from this specific aim is that metabolomics methods and protocols were completed using a C. albicans model system, as described in previous reports. However, it should be noted that spin-off research has evolved from this work, which consists of developing multi-targeted antifungals using phenols abundant in beans and many natural product waste lines. Therefore, another key outcome from this preliminary research is that dietary agents, which have been linked to strengthen our immune system, are also able to remediate or prevent the virulence of an organism responsible for causing 30-40% rates upon infection. Our approach is unique in that dietary phenols present in dry edible beans (ferulic acid, gallic acid, and sinapic) are initially being identified based on their ability to remediate / prevent the non-virulent C.albicans phenotype (yeast) from transitioning into the virulent state (hyphae). At this point in our studies, it has been determined that ferulic acid, gallic acid, and sinapic acid, act together to inhibit the hyphae form, but the effect is dosage and time dependent (data not shown). The second phase of these experiments is to prevent tissue invasion of the hyphal phenotype, another criterion for virulence, via dietary phenolic synergists. Therefore, we are monitoring the ability of several phenols to reduce reactive oxygen species (ROS) that builds in C. albicans as it transitions to the hyphal stage. (The eventual burst of ROS has been linked to tissue invasion.) Our studies have shown that certain phenols are not able to remediate the hyphal phenotype are able to significantly reduce the internal ROSs. Based on this preliminary data, a proposal will be submitted to the newly release NIH RFP-a1-14-066: "Non-Traditional Therapeutics that Limit Antibacterials Resistance (R21/R33)." FTIR and other metabolomics based data that has been accumulated from the methods established from this specific aim are currently being to determine the mechanisms of action of the phenols on C.albicans, particularly on how global metabolism (non-targeted), and central carbohydrate metabolism (targeted) are modulated by the phenols to maintain the non-virulent state. Specific Aim 2: The key outcome from this specific aim is that metabolomics methods and protocols were established that are able to detect subtle and temporal changes in the M1 and M2 phenotype of Raw 264.7 macrophages. These methods included capillary electrophoresis, (specifically for lactic acid, AMP/ATP, and NADH / NAD+) as these intermediates differ between the phenotypes, as M1 exhibits higher levels for other three parameters. Moreover, a FTIR method was also established to allow high throughput analysis between the M1 and M2 state. The data are currently being analyzed. Moreover, colorimetric assays were also set up to measure nitric oxide (NO), which is high in M1 macrophages, but low in M2. Lastly methods were established to determine TCA cycle, nucleotide, amino acid intermediates again using capillary electrophoresis, whereas ion exchange - amperometric chromatography was used to monitor intermediates of glycolysis and the pentose phosphate pathways. (The development of these methods and their application is documented in the dissertation of my former Ph.D. student, and a paper is in preparation.) Using these methods, the ability of phenols to modulate the M1 and M2 state are currently being studied. However, immortal macrophages are being utilized instead of the originally planned bone marrow primary lines due to lack of resources to secure those cells. Still, promising preliminary studies have been obtained from the Raw 264.7 macrophage line using phenols present in beans but as isolated components. First, concentrations were determined that did not affect the viability of the cells were first determined using the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The macrophages were then subjected to lipopolysaccharide, (LPS), i.e., inducer to the M1 state, for 24 hours, followed by treatment (1 ng or 10 ng) with different phenols. Nitric oxide was monitored to determine whether the phenols were able to remediate the bean M1 state back to baseline or possible to the M2 state (data not shown). Nitric oxide was monitored for macrophage exposed to LPS only (inducer of M1), no LPS (inactivated) and Il4 (inducer of M2). Single doses of most of the phenols did not affect the M1 state, with the notable exception of 4-vinyl phenol (1 and 10 ng), catechins (10 ng), and sinapic acid (10 ng). Based upon this data, two phenols were combined and the cells were treated once again in a dose dependent manner (ranging from 0.5:0.5 ng per phenol to 5:5 ng per phenol) after 24 hours post induction of the M1 state with LPS. Combined, the phenols were able to reduce the M1 phenotype to baseline at much lower concentrations with 5 ng of kaempferol and 5 ng protocatechin remediating M1 to baseline (data not showne). Interesting, these two phenols were not able to substantially modulate the M1 used as isolated components at 10 ng each. Moreover, kaempferol-3-glycoside (a major flavonoid in pinto beans) was even able to modulate the M1 phenotype to a M2 state, for at least some of the cells, when combined with pyrogallol. However, 100% of the cells were modulated from the M1 state to the M2 state with 5 pyrogallol was combined wiht farnesol, i.e., a small molecule with not present in beans, but naturally occuring in other natural systems. Specific Aim 3: Specific Aim has not been started yet, due to ability to obtain resources to support the primary cell work. Multiple grant proposal are in preparation to enable completion of this specific aim, including NIH R21 Exploratory grants, American Heart Association grants, AFRI-USDA grants, and internal multistate grants.
Publications
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: Candida albicans have been used to as the model systems to continue to establish metabolomic methods (non-targeted methods (FTIR, direct infusion mass spectrometry) and targeted methods for central carbon metabolism, (capillary electrophoresis, and HPLC)for monitoring the phenolic switch. Therefore, the transition of this switch (from yeast cells to hyphae) has been and is currently being montored with these methods with and without treatments of isolated bioactives and extracts from beans. Moreover, these methods are now being applied to Raw 264.7 macrophages to monitor the switch between the M1 and M2 state, which are currently be activated by lipopolysaccaride and IL-4, respectively. Specific Aim 1: To develop non-targeted and targeted metabolomic approaches capable of detecting subtle and temporal changes in C. albicans phenotypes. The accessed need for Specific Aim 1 is that methods for monitoring the phenotype switches of C. albicans are required to initially develop reliable and rugged metabolomic methods applicable to macrophage phenotypes, i.e., a more complex and costly system. Therefore, Specific Aims 1 and 2 are currently in progress, which are shown below. Specific Aim 1: To develop non-targeted and targeted metabolomic approaches capable of detecting subtle and temporal changes in C. albicans phenotypes. Specific Aim 2: To develop non-targeted and targeted metabolomic approaches capable of detecting potential subtle and temporal changes in the M1 and M2 phenotypes of bone marrow derived macrophages. Specific Aim 3: To determine the ability of pinto beans to modulate the metabolome of M1 phenotype of bone marrow derived macrophages towards the M2 phenotype. PARTICIPANTS: Three graduate students have been assigned to this projects. Collaborations with the School of Biological Sciences (UNL) and the University of Connecticut are in progress to provide assistance and consultation on the C.albicans, and macrophage system, respectively. TARGET AUDIENCES: Target audiences include producers / distributors, / consumers of pinto beans as well as researchers studying phenotypic switches as they apply to any organism, but particularly for C. albicans as an infectious pathogen and macrophages as facilitators of diseased states. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The analytical methods describe above will be used to ultimately determine the effects of diatary components in dry edible beans ability to modulate macrophage phenotypic switches from one of a desease stressing state (M1) to that of a repair state (M2) in order to stop the self-perpetuating macrophage induced chronic inflammation.
Publications
- No publications reported this period
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