Progress 01/29/15 to 09/30/19

Outputs
Target Audience:The research community was the primary target audience for this project. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Postdoctoral fellow Saravanan Devendran led efforts to develop the MBRAs and contributed to the in vitro characterization of bacterial bile acid metabolism described in the papers reported. Postdoctoral fellow Patricia Wolf led the study described that tested the utility of the MBRAs for recapitulating results from prior work in stool bioreactors and contributed to the studies on bacterial bile acid metabolism described in the papers reported. Doctoral student Heidi Doden contributed to the studies on bacterial bile acid metabolism described in the papers reported. How have the results been disseminated to communities of interest?Related studies have been disseminated to the research community through peer-reviewed journal publications. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Mini Bio Reactor Arrays (MBRAs) were successfully built and tested under two different conditions mimicking a meat and vegetable-based diet in order to show that these MBRAs are capable of recapitulating the results seen in prior work in stool bioreactors. Relative abundances from the inoculate of the MBRAs were calculated. As expected, stool inoculate exhibited higher species diversity than either of the MBRA conditions tested. Highly abundant microbial taxa were captured by the MBRAs and the vegetable-based dietary mimic retained more low abundance species than the meat-based diet. These observations provide preliminary validation of the ability of the MBRA to be a high-throughput replacement for classic bioreactor experiments.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Gomez A, Sharma AK, Mallott EK, Petrzelkova KJ, Jost Robinson CA, Yeoman CJ, Carbonero F, Pafco B, Rothman JM, Ulanov A, Vlckova K, Amato KR, Schnorr SL, Dominy NJ, Modry D, Todd A, Torralba M, Nelson KE, Burns MB, Blekhman R, Remis M, Stumpf RM, Wilson BA, Gaskins HR, Garber PA, White BA, Leigh SR. 2019. Plasticity in the human gut microbiome defies evolutionary constraints. mSphere. 2019 Jul 31;4(4). pii: e00271-19. doi: 10.1128/mSphere.00271-19.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Ridlon JM, Devendran S, Alves JM, Doden H, Wolf PG, Pereira GV, Ly L, Volland A, Takei H, Nittono H, Murai T, Kurosawa T, Chlipala GE, Green SJ, Hernandez AG, Fields CJ, Wright CL, Kakiyama G, Cann I, Kashyap P, McCracken V, Gaskins HR. 2019. The 'in vivo lifestyle' of bile acid 7alpha-dehydroxylating bacteria: Comparative genomics, metatranscriptomic, and bile acid metabolomics analysis of a defined microbial community in gnotobiotic mice. Gut Microbes. 2019 Jun 9:1-24. doi: 10.1080/19490976.2019.1618173.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Devendran S, Shrestha R, Alves JMP, Wolf PG, Ly L, Hernandez AG, M�ndez-Garc�a C, Inboden A, Wiley J, Paul O, Allen A, Springer E, Wright CL, Fields CJ, Daniel SL, Ridlon JM. 2019. Clostridium scindens ATCC 35704: Integration of nutritional requirements, the complete genome sequence, and global transcriptional responses to bile acids. Appl Environ Microbiol. 2019 Mar 22;85(7). pii: e00052-19. doi: 10.1128/AEM.00052-19.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Members of the target audience included the research community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Postdoctoral fellow Saravanan Devendran has led efforts to develop the MBRAs (Obj. 1) and contributed to the in vitro characterization of bacterial bile acid metabolism described in the three papers reported. Doctoral student Patricia Wolf contributed to the studies described in Devendran et al., (submitted) manuscript. How have the results been disseminated to communities of interest?Via the publications described. What do you plan to do during the next reporting period to accomplish the goals?We will continue with the original project objectives.

Impacts
What was accomplished under these goals? Efforts have focused on designing and building MiniBioReactor Arrays (MBRAs) as a tool for studying microbial sulfur metabolism in vitro. We are modifying MBRAs described in Auchtung et al. Chapter 18; Adam P. Roberts and Peter Mullany (eds.), Clostridium difficile: Methods and Protocols, Methods in Molecular Biology, vol. 1476, DOI 10.1007/978-1-4939-6361-4_18. A mini bioreactor strip was designed using computer-assisted design (CAD) software and fabricated with DSM Somos Watershed XC 11122 resin via stereolithography (FineLine prototyping). Each MBRA consisted of two reactors, with an internal volume of 25 ml and a working volume of 15 ml. Dimension of the strip is 150mm x 47mm x 36mm. Reactors were drawn with 25 x 25 x 40 mm dimensions, including 10 mm radial blends on the bottom corners and 5 mm radial blends on the top. Reactors were spaced 90 mm center to center to match dimensions on the stir plate. Three 5.55 mm diameter holes (influent, effluent, sampling) were placed into the top of each reactor and spaced 16 mm apart center to center. Inner walls of the reactors were placed 2 mm from the bottom of the strip, 5 mm from the top of the strip, 5 mm from the side of the strip, and 3.25 mm into the strip. The next step is toassemble mini bioreactor strips with tube connections, which will be accomplished as soon as a newlyinstalled anaerobic chamber is fully operational. The studies of Objectives 2 & 3 will then commence. Related efforts have focused on characterizing bacteria strains and genes involved in bile acid metabolism. These bacterial strains will be used in the CCS as an initial means to validate the system justifying the in vitro work. For example, work in the Dolan et al., 2018 paper focused on characterizing 12-alpha hydroxysteroid dehydrogenase (HSDH) activity in strains of Clostridium scindens, Clostridium hylemonae, and Clostridium hiranonis. Future reengineering of 12α-HSDH enzymes to preferentially oxidize cholic acid may provide a means to industrially produce the therapeutic bile acid ursodeoxycholic acid. In addition, a cholic acid-specific 12α-HSDH expressed in the gut may be useful for the reduction in deoxycholic acid concentration, a bile acid implicated in cancers of the gastrointestinal (GI) tract. The research described in Harris et al., 2018 demonstrates that Eggerthella lenta is an important bile acid metabolizing gut microbe and that the gas atmosphere may be an important and overlooked regulator of bile acid metabolism in the gut. Finally, the studies reported in Devendran et al., (submitted) focused on characterizing the basic nutrition and physiology of Clostridium scindens ATCC 35704, one of the major bile acid 7alpha-dehydroxylating anaerobes in the human gut. Pathway analysis of the genome sequence coupled with RNA-Seq analysis of gene expression in defined medium revealed consistency with the growth requirements and end products of metabolism. Induction with bile acids revealed complex and differential responses to cholic acid and deoxycholic acid, including the expression of potentially novel bile acidinducible genes involved in cholic acid metabolism. Responses to toxic deoxycholic acid involved DNA repair, oxidative stress, cell wall maintenance/metabolism, chaperone expression, and down-regulation of one third of the genome. Understanding the metabolic profile of C. scindens ATCC 35704 may help in treatment of disease associated with increased colonic secondary bile acids such as colorectal cancer.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Harris, S.C., Devendran, S., M�ndez-Garcia, C., Mythen, S.M., Wright, C.L., Fields, C.J., Hernandez, A.G., Cann, I., Hylemon, P.B. and Ridlon, J.M. 2018. Bile Acid Oxidation by Eggerthella Lenta Strains C592 and DSM 2243T. Gut Microbes. 2018 Nov 2;9(6):523-539.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Doden, H., Sallam, L.A., Devendran, S., Ly, L., Doden, G., Daniel, S.L., Alves, J.M.P. and Ridlon, J.M. 2018. Metabolism of Oxo-Bile Acids and Characterization of Recombinant 12alpha-Hydroxysteroid Dehydrogenases from Bile Acid 7alpha-Dehydroxylating Human Gut Bacteria. Appl Environ Microbiol. 2018 May 1;84(10).
• Type: Journal Articles Status: Submitted Year Published: 2019 Citation: Devendran et al. 2019. Transcriptional Responses of Clostridium Scindens ATCC 35704 to Bile Acids Under Defined Culture Conditions. Appl Environ Microbiol. (Submitted).

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:The research community was the primary target audience of this project. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported 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?We will continue with the original project objectives.

Impacts
What was accomplished under these goals? Since the project began this past October, efforts have focused on designing and building MiniBioReactor Arrays (MBRAs) as a tool for studying microbial sulfur metabolism in vitro. We are modifyingMBRAs described inAuchtung et al. Chapter 18;Adam P. Roberts and Peter Mullany (eds.), Clostridium difficile: Methods and Protocols, Methods in Molecular Biology, vol. 1476,DOI 10.1007/978-1-4939-6361-4_18 to accomodate a larger volume of medium. We have obtained the CAD files forstereolithography from the corresponding author of the book chapter cited above. Once completed, theMBRAs will be seeded with human fecal microbiota and various sulfur substrates added as described in the Hatch project. Upon satisfactory validation of microbial growth and sulfide production, engineered platforms harboringhuman colonic HCT116 cells will be incorporated downstream of theMBRAsfor completion of Objective 2.

Publications

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Members of the research community were the primary target audience. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The work constitutes a significant part of the doctoral research for a graduate student in the Division of Nutritional Sciences as well as an honor's project for an undergraduate in the Deptartment of Animal Sciences at the University of Illinois. 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?Define the relationship between compartmentalized glutathione redox poise and modes of invasion. Glioma, like many solid tumors, is characterized by a hypoxic core which forces selection of cells capable of altering their metabolic profiles. What is less clear is the extent to which this metabolic adaptation selects for populations capable of aggressive migration. We contend the capability to switch between two modes of movement - amoeboid (rounded, adhesion independent) via activated RhoA or mesenchymal (elongated, adhesion, and proteolysis dependent) via activated Rac1 - allows invasive GBM cells to adapt to varying microenvironments across the margins. We hypothesize intracellular glutathione redox poise serves as a sensor of mechanochemical features across the margins, regulating the ability of GBM cells to switch between elongated and rounded modes of movement required to invade into the brain parenchyma.

Impacts
What was accomplished under these goals? Efforts focused on use of our mitochondrial glutathione biosensors to replicate and extend data provided by Co-Investigator Bartholomew (UIC) regarding the effects of laser illumination on mitochondrial redox homeostasis (Aim 2). That work failed to generate compelling data though it enabled us to become more proficient with the use and interpretation of data from the mitochondrial glutathione biosensors. Accordingly, we decided to not pursue off-site animal work (Aim 1) that would command the bulk of the budget. Instead during the past year, we shifted focus to another emerging collaboration with Professor Brendan Harley in Chemical and Biomolecular Engineering and Dr. Jann Sarkaria at Mayo Clinic. This work focuses on glioblastoma (GBM), the most common, aggressive, and deadly form of brain cancer. Similar to the original proposal, the new project represents a multidisciplinary convergence of our expertise in cell metabolism with a bioengineer and a physician scientist. The GBM efforts have been decidedly more successful and enabled the generation of preliminary data that have been included federal grant applications. Ongoing studies are underway to gather additional data for preparation of manuscripts describing the refocus on role of mitochondrial glutathione in invasive migration of glioblastoma.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2016 Citation: V.L. Kolossov, N. Ponnuraj, J.N. Beaudoin, M.T. Leslie, P.J. Kenis and H.R. Gaskins. 2016. Distinct responses of compartmentalized glutathione redox potentials to pharmacologic quinones targeting NQO1. Biochemical and Biophysical Research Communications doi:10.1016/j.bbrc.2016.12.082.

Progress 01/29/15 to 09/30/15

Outputs
Target Audience:Members of the target audience include the research community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported 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?Aim 1: Use gadolinium-loaded liposomes in magnetic resonance (MR) imaging to quantify the extent to which low level laser therapy (LLLT) and MSC therapy mitigate microvascular rarefaction in a mouse fibrotic kidney model initiated by unilateral ureter obstruction (UUO). We will conduct experiments with either the I/R or UUO mouse model or both in the next funding period. Aim 2: Define the effects of laser illumination wavelengths in the visual light spectrum [red, 635 nm; green, 532 nm; violet, 405 nm] on mitochondrial redox homeostasis in renal epithelial and endothelial cells and mesenchymal stem cells. This work will be extended to additional cell types under varying metabolic conditions in the next funding period.

Impacts
What was accomplished under these goals? Aim 1: Use gadolinium-loaded liposomes in magnetic resonance (MR) imaging to quantify the extent to which low level laser therapy (LLLT) and MSC therapy mitigate microvascular rarefaction in a mouse fibrotic kidney model initiated by unilateral ureter obstruction (UUO). Together with Dr. Sanjay Misra, Mayo Clinic, Co-Investigator Kong has validated a mouse model of renal ischemia/reperfusion (I/R) injury that represents an alternative to the unilateral ureter obstruction (UUO) model of renal failure. Aim 2: Define the effects of laser illumination wavelengths in the visual light spectrum [red, 635 nm; green, 532 nm; violet, 405 nm] on mitochondrial redox homeostasis in renal epithelial and endothelial cells and mesenchymal stem cells. We derived two stable cell lines with mouse embryonic endothelial C166 cells with one harboring the Grx1-roGFP2 sensor targeted to mitochondria and the other the cytosol. A series of studies revealed that 1) neither continuous nor interrupted exposure of C166 cells to 650 nm laser altered basal cytosolic and mitochondrial EGSH; and 2) depletion of GSH with BSO oxidized mitochondrial matrix of C166 cells as expected and additionally rendered cells sensitive to 532 nm laser but did not alter the insensitivity to 650 nm laser.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Kolossov, V.L., Beaudoin, J.N., Ponnuraj, N., DiLiberto, S.J., Hanafin, W.P., Kenis, P.J. and Gaskins, H.R. 2015. Thiol-based antioxidants elicit mitochondrial oxidation via respiratory complex III. Am. J. Physiol. Cell. Physiol. 2015 Jul 15; 309(2):C81-91. PMID: 25994788.