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Changes in the Gut Metabolic Landscape Drive Inflammation-Associated Dysbiosis and Host Responses
Get PDFThe file named "HUGHES-PRIMARY-2022-1.pdf" is the primary dissertation file. Three (3) supplemental files are also available and are referenced in the dissertation file as appendices; the files are Microsoft Excel documents and may be viewed individually.Intestinal inflammation is frequently associated with alterations in composition of gut microbial communities, termed dysbiosis. Inflammation-associated dysbiosis is characterized by an expansion of facultative anaerobic bacteria in the Proteobacteria phylum, such as Escherichia coli. A dysbiotic microbiota has been linked to increased disease severity in the context of inflammatory bowel disease. However, the mechanisms responsible for inflammation-associated dysbiosis and its impact on disease are incompletely understood.
Utilizing bioinformatic analyses of gut microbiota composition and mechanistic studies with Escherichia coli as a model organism in murine models, we uncovered two metabolic pathways that are unique to intestinal inflammation and responsible for changes in microbiota composition. Aerobic respiration coupled with formate oxidation, and utilization of molecular hydrogen fuel expansion of Escherichia coli populations during intestinal inflammation.
The impact of oxygen leakage into the gut lumen on obligate anaerobic bacterial metabolism was additionally investigated. In vitro metabolite measurements and use of bacterial genetics indicated formate production increased in Bacteroides exposed to low oxygen levels. Formate measurements and exogenous delivery of formate in mice suggested that intestinal formate levels increase during inflammation and may exacerbate disease. However, further study is required.
In conclusion, we identified key changes that occur during non-infectious inflammation in the gut metabolic landscape, illustrating the importance of understanding bacterial metabolism in order to understand host-microbiota interactions
The Efficacy of Immunotherapy in Preventing Liver Cancer and the Role of Metabolic Zonation in Its Development
Get PDFIn order to study the development and progression of liver cancer, as well as the efficacy of novel therapeutic strategies, accurate models of human disease are needed. In particular, in vivo mouse models capture critical characteristics that are relevant to human hepatocellular carcinoma (HCC). There is a diverse array of mouse HCC models available, falling into three major categories: transplantation-based models, chemically-induced models, and genetically-induced models. Within these categories, models differ in factors such as the source of the tumor cells and the chemicals or genetic drivers used to induce tumorigenesis. All of these models offer specific advantages over the others but also have some disadvantages as well. Thus, the utility of any model is dependent on the specific investigatory aims of the study. In our case, we used two types of models to try to address two questions about HCC.
First, we asked if immune checkpoint inhibition could prevent tumorigenesis in a chemically-induced mouse model of HCC. We found that initiation of anti-PD-1 immunotherapy prior to tumorigenesis could prevent up to 46% of liver tumors. This reduction in tumor burden was accompanied by infiltration of CD4+ T helper and CD8+ cytotoxic T cells into the liver parenchyma. Importantly, anti-PD-1 therapy did not exacerbate liver dysfunction or worsen overall health in this model. Given the safety and preservation of quality of life observed with long-term immunotherapy use, an immunotherapy chemoprevention strategy is likely associated with a low risk-to-benefit ratio and high value care in select patients.
Along the portal-to-central axis within the hepatic lobule, there are profound differences in gene expression, metabolic processes, oxygen tension, and ploidy. Whether or not these differences reflect any differences in neoplastic potential is unclear. To address this, we turned to genetically-induced HCC models. We used various hepatic zone-specific CreERT2 mouse lines to induce activating mutations in Ctnnb1 and to delete Arid2. We found that mutant clones arising from zone 1 gradually expanded and persisted, while mutant clones arising from Zone 3 rapidly disappeared over time. However, more tumors ultimately developed in the zone 3 livers than in the zone 1 livers, suggesting that expression of some zonated metabolic genes may influence the fate of mutant hepatocytes. This could have major implications for prevention and treatment of HCC, as these metabolic genes could represent actionable preventive or therapeutic targets
Ethical conundrums of current U.S. funding agency security efforts
Get PDFTuesday, November 14, 2023; noon to 1 p.m. (Central Time); Room NB2.100A or via Zoom. "Ethical Conundrums of Current U.S. Funding Agency Security Efforts." Stacy Pritt, D.V.M., M.B.A., Associate Vice President of Research Support and Regulatory Management, UT Southwestern Medical Center.Note: The first 5 minutes of the lecture are not included in the recording because technical difficulties were experienced.Research security became a focal point for U.S. federal agencies funding academic research in 2018 when NIH started sending notifications to dozens of academic institutions about concerns involving foreign influence in research. The effort to determine the scope and extent of the foreign influence, which started years before 2018, continues today and has culminated in the release of draft Research Security Program (RSP) requirements. Lost in this effort are the ethical conundrums presented by the federal government's shifting requirements, inconsistent responses by academic institutions, and the potential chilling effect that all of this has on international research collaborations. This session will break down the concerns U.S. agencies have with inappropriate foreign influence in research, review how those concerns have evolved since 2018, and identify the ethical conundrums inherent to this topic.UT Southwestern--Program in Ethic
Functional Studies of Stem Cells in the Parasitic Flatworm Schistosoma mansoni
No full textSchistosomes are parasitic flatworms that infect over 200 million people, primarily in poverty-stricken developing countries, causing extensive morbidity and mortality. Schistosomes have a great deal in common with other parasitic flatworms such as liver flukes and tapeworms. Namely, they are characterized by a syncytial "skin" known as the tegument that is critical for their survival within their hosts, they possess somatic stem cells referred to as neoblasts, and they are incredibly successful parasites. Until now, there has been little appreciation for the interrelationships between the tegument, neoblasts, and successful parasitism.
Our recent work, however, suggested that schistosome neoblasts give rise to cells that are associated with the parasite's tegument. In order to determine whether schistosome neoblasts produced the tegument, we developed novel labeling techniques and found that neoblasts give rise to progenitor cells that ultimately maintain the tegument. We also developed a fluorescence activated cell sorting protocol that we used to isolate neoblasts and tegument progenitors and obtain their transcriptomes. With this transcriptome data, we identified two regulators of tegument maintenance, zfp-1 and zfp-1-1, that are functionally conserved in free-living flatworms, suggesting a common molecular program for "skin" production in all flatworms. Additionally, our work suggested that zfp-1-1 specifically and tegument maintenance generally may be valid therapeutic targets.
We next wondered whether schistosome neoblasts were responsible for making or maintaining any non-tegumental tissues in the adult parasite. To study this, we employed single cell RNAseq to create a gene expression atlas of 43,642 cells from adult parasites. This atlas gave us molecular markers for 68 distinct clusters of cells ranging from muscles and neurons to reproductive tissues such as the ovary. It also allowed us to identify a previously unknown sub-population of neoblasts that appears to exist at the top of a schistosome gut lineage. In an effort to study these neoblasts, we found that the schistosome homolog of the nuclear receptor hnf4 is required for normal gut homeostasis and that loss-of-function of hnf4 prevents parasite blood feeding and abrogates disease pathology in vivo.
Taken together, this work demonstrates how studying basic developmental processes (i.e. stem cell differentiation) in a pathogenic organism can lead to not only insights into evolutionary biology (the machinery that regulates skin production appears to be conserved across flatworms), but it can also suggest novel therapeutic targets (namely zfp-1-1 and tegument maintenance as well as hnf4 and gut homeostasis)
Structure and Function of the Alpha3Beta4 Nicotinic Acetylcholine Receptor
No full textNicotinic acetylcholine receptors are pentameric ligand-gated ion channels that are essential for the proper function of the central and peripheral nervous systems. The α3β4 subtype is highly expressed in the autonomic ganglia, where it contributes to signal transduction from the central nervous system to the periphery. Moreover, α3β4 receptors are found in key brain regions that modulate reward circuits and have therefore been identified as potential targets for anti-addiction therapeutics. Given the physiological importance of this protein, I sought to understand the molecular mechanisms underlying ligand recognition, channel gating, and ion permeation in the α3β4 nicotinic acetylcholine receptor.
Paramount to this goal was the pursuit of a high-resolution structure of the α3β4 subtype. I initially attempted to determine a crystal structure of this receptor before taking advantage of recent technological advances in cryo-electron microscopy. Using this method, I solved the first structure of the α3β4 nicotinic receptor, which was also the first high-resolution structure of any nicotinic acetylcholine receptor in a lipidic environment. By obtaining structural information of the protein bound to a non-selective nicotinic agonist as well as an α3β4-selective ligand, I was able to draw conclusions regarding ligand-selectivity in the nicotinic receptor family. Furthermore, these structures provided a detailed view of the ordered regions of the intracellular domain for the first time, giving insight into the full ion permeation pathway of these channels. This work also provided a blueprint to examine other outstanding questions in the field. Specifically, I used structural and functional approaches to begin to understand the consequences of accessory subunit incorporation, the role of multivalent cations in the desensitization of nicotinic receptors, and the role of the intracellular domain in ion selectivity and rectification
Biochemical Characterization of IpaH E3 Ubiquitin Ligase Effector Proteins and Their Host Substrates
Get PDFShigella flexneri is a gram negative pathogen that utilizes its type 3 secretion system (T3SS) to inject effector proteins in the cytoplasm of host cells to manipulate host cells processes. T3SS effectors are able to post translationally modify host proteins to reprogram intracellular signaling pathways, actin dynamics, membrane trafficking, and innate immune pathways. This allows Shigella to modify the intracellular environment to be conducive to bacterial replication and dissemination to neighboring cells. Shigella flexneri and other bacteria including Salmonella and Yersinia secreted E3 ubiquitin ligases into the host cell cytoplasm via the Type III secretion system (T3SS) apparatus. The invasion plasmid antigen Hs (IpaHs) are a novel family of bacterial E3 ubiquitin ligases that are secreted by Shigella, Salmonella, and Yersinia. These bacterial enzymes highjack the host ubiquitin conjugation machinery by binding to ubiquitin-charged E2 conjugating enzymes and facilitating direct transfer of ubiquitin onto host substrates. IpaH effectors induce polyubiquitination and subsequent proteasomal degradation of their substrates during bacterial infection. The effector substrate interaction of IpaH1.4/2/5 and HOIP was previously characterized. I went on to identify that IpaH2.5 is able to inhibit the in vitro catalytic activity of HOIP via mono-ubiquitination of catalytic lysine residues in the HOIP ring-between-ring domain (RBR-C). Subsequent to this Ubiquitin activated interactive trapping (UBAIT) screening was then utilized to identify the host substrate of IpaH7.8, Gasdermin B (GSDMB). GSDMB belongs to a large family of pore forming cytolysins that execute inflammatory cell death programs. While genetic studies have linked GSDMB polymorphisms to inflammatory disease, its function in human physiology remains poorly understood. I investigated a previously unrecognized host-pathogen conflict between GSDMB and the IpaH7.8 effector protein encoded by Shigella flexneri. Through extensive biochemical and cellular characterization, I show that IpaH7.8 ubiquitinates and targets GSDMB for proteasome destruction. This virulence strategy protects Shigella from the bacteriocidic activity of Natural Killer cells by suppressing Granzyme-A mediated activation of GSDMB. In contrast to the canonical function of most Gasdermin-family members, GSDMB does not inhibit Shigella by lysing infected cells. Rather, GSDMB exhibits direct microbiocidal activity through recognition of phospholipids found on Gram-negative bacterial membranes. These findings place GSDMB as a central executioner of intracellular bacterial killing and reveals a mechanism employed by pathogens to counteract this host defense system
Academic hospital medicine: the good, the bad and the lovely
Get PDFDetailed formal protocol with illustrations and extensive bibliography.A recording of the protocol presentation is available on UT Southwestern's Mediasite. Note: Access to the video is restricted to authorized UT Southwestern users only.UT Southwestern--Internal Medicin
Dignity in later life (The Daniel W. Foster, M.D., Visiting Lectureship in Medical Ethics)
Get PDFThe Daniel W. Foster, M.D. Visiting Lectureship In Medical Ethics (in conjunction with Ethics Grand Rounds). Tuesday, December 12, 2023; noon to 1 p.m. (Central Time); Room NB2.100A or via Zoom. "Dignity in Later Life." Nancy S. Jecker, Ph.D., Professor of Bioethics and Humanities at the University of Washington School of Medicine.Some argue dignity is a useless concept that bioethics can do without. Against this view, I show dignity is a central concern for bioethics, particularly for older people. Dignity's importance during later life is part of a broader view I call the life stage relativity of values. It holds that different values emerge as central at different periods of our lives. During early life, caring, trust, and nurturing figure prominently due to vulnerabilities that characterize infancy and childhood. By adulthood, greater physical and emotional independence leads to autonomy and self-reliance taking center stage. During later life, heightened risk for chronic disease and disability makes keeping dignity intact a critical concern. Across the lifespan, the highest value for an individual relates to their life stage circumstances. Ignoring this can lead to life stage bias, especially midlife bias, which occurs when we apply values central during midlife to all life stages.UT Southwestern--Program in Ethic
Bone Health Outcomes in Post-Lung Transplant Patients with Cystic Fibrosis
Get PDFBACKGROUND: Osteoporosis is a common comorbidity in patients with cystic fibrosis (CF). Although lung transplantation (LTx) improves quality of life of CF patients, there is little research examining long-term bone health outcomes following LTx in these patients.
OBJECTIVE: We sought to compare long-term bone health outcomes in LTx patients with and without CF, as well as determine factors associated with adverse bone health in CF patients.
METHODS: Data were collected on 59 patients who underwent LTx between 2006-2019, including 30 with CF and 29 without CF. We compared baseline characteristics, long-term bone mineral density (BMD) trends, and fracture incidence between the two patient populations, and examined factors associated with post-LTx fractures in CF patients.
RESULTS: Compared with non-CF patients, patients with CF were younger, had lower body mass index, and lower baseline BMD Z-scores at the lumbar spine, femoral neck, and total hip (all p<0.001). BMD at all sites declined in both groups in the first year post-LTx. In subsequent years, CF patients exhibited better BMD recovery relative to pre-transplantation, but continued to have lower BMD post-LTx. Post-transplant fractures occurred in 30% and 34% of CF and non-CF patients, respectively. CF patients who developed fractures after LTx had significantly lower BMD and lower pre-transplantation percent predicted forced expiratory volume in one second (FEV1%).
CONCLUSION: Although CF patients exhibit better BMD recovery following LTx compared to their non-CF counterparts, CF patients start with significantly lower pre-LTx BMD and experience a similarly high rate of post-LTx fractures. These findings highlight the unique contribution of the CF disease process to bone health, as well as a clear need for better prevention and treatment of osteoporosis in CF patients before and after LTx
Development of Video and Simulation-Based Communication Skills Learning: Responding to Emotions
No full textThe general metadata -- e.g., title, author, abstract, subject headings, etc. -- is publicly available, but access to the submitted files is restricted to UT Southwestern campus access and/or authorized UT Southwestern users.PROBLEM: Responding appropriately to patients’ emotions and concerns is vital for excellent patient care and outcomes. There is a lack of training in this area, with the need to educate healthcare providers about how to appropriately respond to emotions. While communication skills training programs exist for undergraduate medical education, medical students often feel unprepared in responding to patients’ emotions
INTERVENTION: A 90-minute small group exercise was developed for second-year pre-clinical students at a large United States medical school in Texas. The exercise consisted of faculty-facilitated small group discussion of two video examples of a patient encounter, comparing clinician responses to patient emotions. The exercise utilized a framework for identifying skills and patient impact of verbal expressions of empathy to different patient emotions.
CONTEXT: This curriculum sought to demonstrate examples of strong patient emotions for preclerkship students, and introduce a framework of concrete communication skills that can positively impact a clinical encounter. Data was collected by questionnaires delivered immediately pre-session and post-session, as well as 3 months post-session. Survey questions assessed student perception of effectiveness of the exercise, student-reported preparedness and feeling equipped to perform various communication skills during clinical visits. Paired t-tests were performed and data analyzed for qualitative responses.
OUTCOMES: The process evaluation yielded a positive subjective learner response to the exercise which was sustained at 3-month follow-up. The students (N=161 paired for the immediate pre- and post-survey) reported significant increases in knowledge and preparedness to recognize and appropriately respond to different patients’ emotions (P= 0.001). Qualitative data were also captured in the surveys.
LESSONS LEARNED: This video-based small group discussion of skills to express verbal empathy is perceived by pre-clinical medical students to be beneficial, and to improve their knowledge and preparedness for using empathic skills in the future. There is a need for further investigation whether this type of communication skills training results in behavior change and is sustained long-term